Multicenter evaluation of a transcription-reverse transcription concerted assay for rapid detection of mycobacterium tuberculosis complex in clinical specimens.

A European multicenter study was performed to evaluate the performance of a new method, based on the transcription-reverse transcription concerted reaction (TRC-2), which enabled one-step amplification and real-time detection of the Mycobacterium tuberculosis 16S rRNA target directly in clinical specimens. A total of 633 respiratory and nonrespiratory specimens were tested, and the results were compared with those from smears and cultures. A total of 129 patients (Paris center) were followed up in order to evaluate the clinical performance of TRC-2. By using M. tuberculosis complex strains to inoculate sterile sputa, the detection limit of TRC-2 was found to be 30 to 50 CFU/ml. A total of 548 respiratory specimens and 59 extrapulmonary specimens were assessable. For pulmonary specimens, the sensitivities of TRC-2 and acid-fast smear were 86.8% and 50.4%, respectively (P = 0.002). The specificities were 97.5% and 100%, respectively. For extrapulmonary specimens, the sensitivities of TRC-2 and acid-fast smear were 83.3% and 8.3% (P < 0.0001), and the specificities were 95.8% and 100%, respectively. Fifteen of 129 patients were diagnosed with pulmonary tuberculosis (TB). The sensitivities of culture and TRC-2 were 80% (12/15) and 86.7% (13/15) (P = 0.16), and the specificities were 100% and 93.9%, respectively. Based on an 11.6% incidence of TB in our population, the positive predictive values of TRC-2 and culture were 81.3% and 100%, respectively, and the negative predictive values were 98.2% and 97.4%, respectively. These results demonstrated that detection of M. tuberculosis complex in clinical specimens by TRC-2 with ready-to-use reagents was an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB.

IFNgamma and antibody responses among French nurses during a tuberculosis contact tracing investigation.

STUDY: A comparative study which compared PPD skin testing inserted according to the French Society of Pneumology's recommendations and interferon gamma release assay (IGRA) (QuantiFERON((R)) TB Gold In-tube, QF-TB-IT, Cellestis, Carnegie, Australia) was performed during a tuberculosis contact investigation in our hospital. PATIENTS: Nineteen French health-care workers (HCWs) volunteered to participate. All of the HCW enrolled were BCG vaccinated and had a normal chest X-ray at entry. RESULTS: Among the HCW, 68.4% were TST positive. By comparison, only 31.6% had a positive QF-TB-IT result. We took advantage of the negative tube and the corresponding plasma for antibody detection by ELISA. None were ELISA positive. Fourteen HCWs were followed up. None of the HCWs accepted a course of antiTB chemoprophylaxis. Despite the difficulty in establishing a trend in kinetics, we saw the complexity of interpretation of a dynamic T-cell response after contact with an index case. CONCLUSION: This initial and first French picture provides us with the observation that only 44% of TST-positive HCW were IGRA positive, and the IGRA test allowed the detection of LTBI in two TST negative HCWs.

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.

[New immunological tests in the diagnosis of tuberculosis]. / Les nouveaux tests immunologiques dans le diagnostic de la tuberculose (TB or not TB).

INTRODUCTION: Targeted testing and treatment of individuals with latent tuberculosis infection (LTBI), at high risk of progression to active tuberculosis (ATB), are key elements in the battle against tuberculosis, both in France and in many parts of the world. Though the finding of tubercle bacilli is the essential examination for the diagnosis of ATB, there is no indisputable test for LTBI. BACKGROUND: The help currently given to the diagnosis of LTBI by the degree of positivity of the tuberculin skin test (TST) is limited, both operationally and logistically, in populations vaccinated with BCG or sensitised by atypical mycobacteria, and by its low sensitivity in those immuno-suppressed persons who are at greatest risk of progression. Moreover the TST has other operational limitations linked to return visits, repeat testing causing a boosting effect and subjective interpretation. A new approach follows the availability of two biological tests for the diagnosis of LTBI (QuantiFERON-TB and T-SPOT-TB) that measure the in-vitro production of interferon gamma (IFN-gamma) by the blood mononuclear cells in response to M. tuberculosis specific antigens (ESAT-6 and CFP10). This revue analyses the published studies, undertaken with varying numbers of patients, that evaluate the diagnostic accuracy of these two tests in comparison with TST. However, validation is handicapped by the lack of a "gold standard" for the diagnosis of LTBI. These studies demonstrate similar levels of specificity for the two biological tests. They are statistically higher than those for TST, particularly in populations vaccinated by BCG. On the other hand, their sensitivity was at least equivalent to that of TST and, in certain studies, superior with T-SPOT-TB. Finally, several studies in contacts have been undertaken with the aim of measuring the concordance between these biological tests and TST. The essential finding is of a very good correlation between positivity of the biological tests and the degree of exposure of the contacts. These tests have additional operational advantages over TST: completed in one visit, results available in 24 hours, absence of inter and intra observer divergence, detection of potential immuno-depression and avoidance of boosting by repeat testing. VIEWPOINT: Currently, however, these biological tests present several operational limits: lower sensitivity in severe disease, incomplete data in immuno-suppressed subjects and in children, lack of predictive value for future development of ATB, lack of distinction between LTBI and ATB. Numerous clinical studies are under way, in France and elsewhere, in order to reduce these limitations and to allow the appropriate incorporation of these tests into protocols for the diagnosis of tuberculosis. CONCLUSIONS: These two biological tests should, in the near future, replace or complement TST in the diagnosis of recent LTBI, leading to their optimal incorporation into the decision making processes of the national plans for the control of tuberculosis.

Enhancement of superconductivity and evidence of structural instability in intercalated graphite CaC6 under high pressure.

We measured the temperature dependent resistivity, varrho(T), of the intercalated graphite superconductor CaC6 as a function of pressure up to 16 GPa. We found a large linear increase of critical temperature, Tc, from the ambient pressure value 11.5 K up to 15.1 K, the largest value for intercalated graphite, at 7.5 GPa. At approximately 8 GPa, a jump of varrho and a sudden drop of Tc down to approximately 5 K indicates the occurrence of a phase transition. Our data analysis suggests that a pressure-induced phonon softening related to an in-plane Ca phonon mode is responsible for the Tc increase and that higher pressures greater, similar8 GPa lead to a structural transition into a new phase with a low Tc less, similar3 K.

[Advantages and drawbacks of in vitro Interferon-gamma/T cell assays compared to the Mantoux test for the diagnosis of tuberculosis]. / Avantages et limites des tests sanguins in vitro lymphocytes T/interféron gamma comparativement au test intradermique à la tuberculine pour le diagnostic de tuberculose.

The development of in vitro blood tests that measure the delayed hypersensitivity reaction developed after contact with Mycobacterium tuberculosis will change progressively the diagnosis of M. tuberculosis infection. These blood assays (Quantiferon TB Gold, Cellestis, Australia; T-SPOT.TB, Oxford Immunotec, United Kingdom) use specific, complex M. tuberculosis antigens (ESAT-6 and CFP-10), whereas the intra-dermal Mantoux test is done with tuberculin, a complex mixture of more than 200 antigens. ESAT-6 and CFP-10 are absent from all the BCG vaccine strains used throughout the world. Significant improvement in the specificity with equivalent or increased sensitivity of the in vitro tests compared to the Mantoux test will lead eventually to replacement of the latter.

[Automated RNA amplification for the rapid identification of Mycobacterium tuberculosis complex in respiratory specimens]. / Identification rapide de Mycobacterium tuberculosis complex dans les échantillons respiratoires par amplification d'ARN en temps réel.

Rapid and sensitive detection of Mycobacterium tuberculosis complex (MTB) directly on clinical respiratory specimens is essential for a correct management of patients suspected of tuberculosis. For this purpose PCR-based kits are available to detect MTB in respiratory specimen but most of them need at least 4 hours to be completed. New methods, based on TRC method (TRC: Transcription Reverse transcription Concerted--TRCRapid M. Tuberculosis--Tosoh Bioscience, Tokyo, Japon) and dedicated monitor have been developed. A new kit (TRC Rapid M. tuberculosis and Real-time monitor TRCRapid-160, Tosoh Corporation, Japan) enabling one step amplification and real-time detection of MTB 16S rRNA by a combination of intercalative dye oxazole yellow-linked DNA probe and isothermal RNA amplification directly on respiratory specimens has been tested in our laboratory. 319 respiratory specimens were tested in this preliminary study and results were compared to smear and culture. Fourteen had a positive culture for MTB. Among theses samples, smear was positive in 11 cases (78.6%) and TRC process was positive in 8 cases (57.1%). Overall sensitivity of TRC compared to smear positive samples is 73%. Theses first results demonstrated that a rapid identification of MTB was possible (less than 2 processing hours for 14 specimens and about 1 hour for 1 specimen) in most cases of smear positive samples using ready to use reagents for real time detection of MTB rRNA in clinical samples. New pretreatment and extraction reagents kits to increase the stability of the sputum RNA and the extraction efficiency are now tested in our laboratory.

Scanning tunneling spectroscopy on the novel superconductor CaC6.

We present scanning tunneling microscopy and spectroscopy of the newly discovered superconductor CaC6. The tunneling conductance spectra, measured between 3 and 15 K, show a clear superconducting gap in the quasiparticle density of states. The gap function extracted from the spectra is in good agreement with the conventional BCS theory with Delta0=1.6+/-0.2 meV. The possibility of gap anisotropy and two-gap superconductivity is also discussed. In a magnetic field, direct imaging of the vortices allows us to deduce a coherence length in the ab plane xiab approximately 33 nm.

[The role of human dendritic cells in tuberculosis: protector or non-protector?]. / Rôle des cellules dendritiques humaines dans la tuberculose: protecteur ou non protecteur?

INTRODUCTION: Mycobacterium tuberculosis, the cause of tuberculosis remains a pathogenic organism capable of infecting a large number of individuals and of resisting the immune response of the infected host. The main constituents of this response are the antigen presenting cells such as dendritic cells, macrophages and T lymphocytes. BACKGROUND: Comparative study of the interactions between M. tuberculosis and the antigen presenting cells has shown that dendritic cells do not permit intracellular growth of M. tuberculosis, unlike that seen in macrophages. A hostile intracellular compartment creates a bacteriostatic environment. M. tuberculosis is internalised by binding to a C-type lectin receptor (DC-SIGN). VIEWPOINT: This receptor recognises polysaccharide compounds on the surface of M. tuberculosis. This sugar-lectin bond may compensate for the bond between bacterial compounds and Toll receptors, partially inhibiting the protective inflammatory reaction or compensating for an excessive inflammatory reaction. CONCLUSIONS: This bond encourages both the persistence of quiescent bacteria in the dendritic cells and the reciprocal adaptation of the host and the bacteria over the course of time.

Experimental evidence of s-wave superconductivity in bulk CaC6.

The temperature dependence of the in-plane magnetic penetration depth, lambda(ab)(T), has been measured in a c-axis oriented polycrystalline CaC(6) bulk sample using a high-resolution mutual inductance technique. A clear exponential behavior of lambda(ab)(T) has been observed at low temperatures, strongly suggesting isotropic s-wave pairing. Data fit using the standard BCS theory yields lambda(ab)(0) = (720 +/- 80) A and delta(0) = (1.79 +/- 0.08) meV. The ratio 2delta(0)/k(B)T(c) = (3.6 +/- 0.2) gives indication for a weakly coupled superconductor.

Superconductivity of bulk CaC6.

We have obtained bulk samples of the graphite intercalation compound, CaC6, by a novel method of synthesis from highly oriented pyrolytic graphite. The crystal structure has been completely determined showing that it is the only member of the MC6, metal-graphite compounds that has rhombohedral symmetry. We have clearly shown the occurrence of superconductivity in the bulk sample at 11.5 K, using magnetization measurements.

[Antiviral vaccination and respiratory mucosal immunity: still disappointing results from a seductive idea]. / Vaccination antivirale et immunité muqueuse respiratoire: un concept séduisant pour des résultats encore décevants.

Mucosal surfaces of the respiratory tract represent a major portal of entry for most human viruses and a critical component of the mammalian immunologic repertoire. The major antibody isotype in external secretions is secretory immunoglobin A (S-IgA). The major effector cells in mucosal surfaces, however, are not IgA B cells, but T lymphocytes, which may account for up to 80% of the mucosal lymphoid cell population. Mucosal immunoprophylaxis is theoretically an important approach to control infections acquired through these portals. Passive antibodies can protect against mucosal viral infections, as shown for respiratory syncytial virus, but very high quantities of passive antibodies are needed to restrict virus replication on mucosal surface. Factors likely to induce mucosal antibody and cell-mediated immune responses include oral or respiratory routes of immunization and active (effectively replicating) vaccine agents. Very few antiviral vaccines have been developed to protect the mucosal surface of the respiratory tract, and only an attenuated influenza virus vaccine uses the nasal route. Other vaccines, approved for parenteral use, have been administered experimentally by the nasal route; these include active (replicating) and inactive (nonreplicating) vaccines. By this route they induce only a moderate local mucosal response. Neither the development of mucosal immunity nor the administration of vaccines via the mucosal route is essential for control or prevention of most respiratory viral infections and diseases acquired through the respiratory tract. Nonetheless, the example of the live attenuated intranasal influenza vaccine, which induces both systemic and local immune response, is promising for the future of mucosal immunization against respiratory viral infections.

[Granulysin: antimicrobial molecule of innate and acquired immunity in human tuberculosis]. / Granulysine: médiateur de l'immunité innée et de l'immunité acquise dans la tuberculose chez l'homme.

The recent global increase in cases of tuberculosis and the emergence of multidrug-resistant strains of tuberculosis have focused attention on the molecular mechanisms of human antimycobacterial immunity. The macrophage is not only the primary site for Mycobacterium tuberculosis growth but also ordinarily provides the primary lines of host defense against invading pathogens in its role as an effector of innate immunity. The ability of M. tuberculosis to survive and replicate in the host macrophage is critical to its pathogenesis, emphasizing a need for a clearer understanding of its interactions with the host macrophage. Macrophages use varied strategies to kill and destroy invading organisms, including production of reactive nitrogen and oxygen intermediates, phagosome maturation and acidification, fusion with lysosomes, exposure to defensins and host cell apoptosis. In human, granulysin is a recently identified antimicrobial protein expressed on cytotoxic T cells, natural killer (NK) cells and NKT cells. It has been shown that granulysin contributes to the defense mechanisms against mycobacterial infection. We hypothesized that human macrophages may possess antimicrobial substances, such as granulysin, and play a role in the defense mechanism.

Evaluation of the automated phoenix system for potential routine use in the clinical microbiology laboratory.

A comparative study was designed to evaluate the identification (ID) and antimicrobial susceptibility testing (AST) performances of the BD Phoenix Automated Microbiology System (Becton Dickinson Diagnostic Systems [BD], Pont de Claix, France). A total of 305 single clinical isolates were collected, and comparisons were made with routine manual methods in use in our microbiology laboratories. The percentages of correct IDs were 93.3, 89.4, 91.8, and 85.7% for enterobacteria, nonfermenting gram-negative bacilli, staphylococci, and streptococci-enterococci, respectively. The median ID time was 3 h, and the median time for AST was 10 h 30 min. AST results showed variable percentages of errors for the different antibiotics. None of the enterobacteria and 0.3% of Pseudomonas aeruginosa isolates showed a very major error (VME). Only one strain of Staphylococcus aureus showed a VME with oxacillin. We demonstrate here the efficiency of the Phoenix system, which can be used for the majority of strains encountered in a university-based laboratory, for ID and AST.

Rapid diagnosis of extrapulmonary tuberculosis by PCR: impact of sample preparation and DNA extraction.

In cases of suspected extrapulmonary tuberculosis, rapid and accurate laboratory diagnosis is of prime importance, since traditional techniques of detecting acid-fast bacilli have limitations. The major difficulty with mycobacteria is achieving optimal cell lysis. Buffers used in commercial kits do not allow this complete lysis in a number of clinical specimens. A comparison of two sample preparation methods, pretreatment with proteinase K (PK-Roche) and complete DNA purification (cetyltrimethylammonium bromide [CTAB]-Roche), was conducted on 144 extrapulmonary specimens collected from 120 patients to evaluate the impact on the Cobas-Amplicor method. Thirty patients were diagnosed with tuberculosis, with 15 patients culture positive for Mycobacterium tuberculosis. Amplification and detection of the amplicons were impaired by a high number of inhibitory specimens (39 to 52%). CTAB-Roche allowed the detection of more culture-positive specimens by PCR than PK-Roche. Comparison with the final diagnoses of tuberculosis confirmed that CTAB-Roche produced the best sensitivity (53.8%) compared to culture (43.3%), PK-Roche (16%), and smear (13%). However, the specificity of the PCR assay with CTAB-Roche-extracted material was always lower (78.8%) than those with culture (100%) and PK-Roche (96.5%). False-positive specimens were lung biopsy material, lymph node biopsy material and aspirate, or bone marrow aspirate, mainly from immunocompromised patients. Despite the efficiency of complete DNA extraction for the rapid diagnosis by PCR of extrapulmonary tuberculosis, the false-positive results challenge our understanding of PCR results.

Trypanosoma cruzi: infection patterns in intact and athymic mice of susceptible and resistant genotypes.

Inbred strains of mice inoculated with the T cruzi Y strain behaved as susceptible (A/J, C3H/HeN), intermediate (BALB/c) or relatively resistant (C57BL/6) with respect to the magnitude of parasitaemia and mortality rate. C57BL/10 mice were susceptible in relation to parasitaemia but resistant when mortality was analyzed. Infection with T cruzi CL strain presented the same results, except for C57BL/6 which behaved as susceptible mice. Athymic mice of various backgrounds revealed no differences in susceptibility, presenting the same dramatic parasitaemia, tissue colonization pattern and no inflammatory reaction in any of the tissues studied. Infection of euthymic and athymic BALB/c mice elicited the production of parasite-specific antibodies, which reached similar levels on the first 9 days but differed after day 13. Serum transfer experiments in BALB/c mice did not show great differences in parasitaemia but altered T. cruzi polymorphism reducing the slender forms in athymic mice. Histopathology of athymic BALB/c mice showed the same tissue tropism when infected either with T cruzi Y or CL strain.