Quinoxaline-based efflux pump inhibitors restore drug susceptibility in drug-resistant nontuberculous mycobacteria.

Nontuberculous mycobacteria (NTM) comprise several ubiquitous, environmentally localized bacteria that may be responsible for serious human diseases. NTM-associated pulmonary infections largely affect individuals with underlying respiratory disease or chronic disease and immunosuppressed patients. Mycobacterium simiae and M. abscessus are two NTMs responsible for lung disease in immunocompetent and immunocompromised individuals. In this study, two NTM strains were isolated from two patients admitted to an Italian hospital and were identified as M. simiae and M. abscessus. The two NTMs were tested for drug susceptibility against different antibiotics. To restore drug susceptibility, a new series of 2-aryl-3-phenoxymethyl-quinoxaline derivatives (QXs) was designed, synthesized, and investigated as efflux pump inhibitors (EPIs) against two clinical isolates of the above-cited NTMs, evaluating how EPIs can influence the drug minimal inhibitory concentration values and, therefore, the activity. The different\ resistance levels tracked in the clinical strains were reduced by EPIs, and in several cases, the susceptibility was completely restored. QXs also resulted as potential chemical probes to be used in drug susceptibility tests to identify the resistance origin when detected.

Effect of teriflunomide on QuantiFERON-TB Gold results.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system characterized by damage to myelin and axons, over time leading to progressive neuronal degeneration and microglial activation. There is still no curative treatment, but during the last 20 years eight different therapies have become available including interferon beta, glatiramer acetate, teriflunomide, dimethyl fumarate, natalizumab, fingolimod, alemtuzumab, mitoxantrone and teriflunomide. Teriflunomide is an immunomodulatory drug that exerts an inhibitory effect on T cell activation in central nervous system of the patients with multiple sclerosis. We determined whether teriflunomide affect the production of interferon-gamma, interleukin-2 and tumor-necrosis-factor-α in the QuantiFERON-TB in-Tube-assay. Blood from 24 adults with latent tuberculosis infection was added to one standard set of QuantiFERON tubes and one further set containing teriflunomide. Teriflunomide resulted in a change in QuantiFERON results from positive to negative in four patients with a marked reduction in interferon-γ. Our data indicated that results from QuantiFERON in patients on teriflunomide therapy should be interpreted with caution.

QuantiFERON-TB Gold Assay on Plasma for Confirmation of Presumed Tuberculosis-Related Uveitis.

The QuantiFERON-TB Gold assay was used to measure interferon gamma levels in plasma from 4 patients with presumed tuberculosis-related uveitis before, during, and after antitubercular therapy. After treatment, all patients showed clinical improvement. The concentrations showed a reversion to an absence of interferon gamma in one case, decreased in two cases, and remained stable in one case. These results suggest that the QuantiFERON assay may be useful for tuberculosis-related uveitis diagnosis and follow-up.

Tuberculin skin test and QuantiFERON in children.

Until some time ago, the tuberculin skin test was the only available screening test for the diagnosis of tubercular infection. Now the new interferon-? release assay QuantiFERON-TB Gold shows promise of greater accuracy in the detection of Mycobacterium tuberculosis-infected subjects. The aim of our study was to evaluate the use of QuantiFERONTB Gold in children and to verify its agreement with the tuberculin skin test. A total of 27 children had a positive tuberculin skin test, 76 subjects were negative and the remaining 2 had a dubious Mantoux test. A positive QuantiFERONTB Gold result was obtained in 21 children while in 84 it was negative. No statistically significant difference was detected between the two assays, which showed a concordance of 90.57%. Our results demonstrated a good concordance between the tuberculin skin test and the interferon-? release assay, though the QuantiFERON-TB may have several advantages over the Mantoux test.

Identification of non-tuberculous mycobacteria from clinical samples.

Non-tuberculous mycobacterial infections cause morbidity worldwide. NTM are considered opportunistic pathogens, and several species have been associated with human disease which has typically pulmonary, skin and soft tissue, lymphatic or disseminated presentation. This study evaluated the distribution of non-tuberculous mycobacteria in Sardinia. Mycobacterium avium, Mycobacterium gordonae and Mycobacterium xenopi were frequently found. Our results agreed with literature data both for the frequent isolation of M. avium, M. xenopi and M. gordonae, and the symptoms and radiological evidence of the patients analysed.

Preliminary data of different methods for the indirect diagnosis of Mycobacterium bovis infection.

We compared the response induced by QuantiFERON-TB Gold antigens to that obtained with the Intradermal Comparative Tuberculin Test and BOVIGAM assay. Our results showed that the QuantiFERON-TB Gold technique used in humans could also be applied for the diagnosis of TB infection in cattle.

Molecular characterization of Sardinian Mycobacterium tuberculosis isolates by IS6110 restriction fragment length polymorphism, MIRU-VNTR and rep-PCR.

An evaluation of the utility of rep PCR typing compared to the 15 loci discriminatory set of MIRU-VNTR was undertaken. Twenty-nine isolates of Mycobacterium tuberculosis from patients were examined. Genomic DNA was extracted from the isolates by standard method. The number of copies of tandem repeats of the 15 MIRU-VNTR loci was determined by PCR amplification and agarose gel electrophoresis of the amplicons. M. tuberculosis outbreak-related strains were distinguished from other isolates. MIRU-VNTR typing identified 4 major clusters of strains. The same isolates clustered together after RFLP typing, but rep-PCR identified only 3 of them. The concordance between RFLP and MIRU-VNTR typing was complete, with the exception of two isolates with identical RFLP patterns that differed in the number of tandem repeat copies at two MIRU-VNTR alleles. A further isolate, even sharing the same RFLP pattern, differed by one repeat from the rest of its cluster. We also tested the use of an automated rep-PCR for clinical laboratory applications but it failed to identify the link between two pairs of epidemiologically related strains clustered by the other 2 techniques. For superior discrimination, ease of comparison of results and lower cost, MIRU-VNTR typing should be the favored PCR-based typing tool.

"In vitro" activity of Melaleuca cajuputi against mycobacterial species.

The increasing incidence of resistance in tuberculosis and in atypical mycobacterial infections has prompted the search for alternative agents. We explored the antimycobacterial activity of Melaleuca cajuputi essential oil against tubercular and non tubercular mycobacterials isolates. The good activity observed towards M. cajuputi indicated that this essential oil might represent a promising antimicrobial agents, particularly in the management of microbial resistance.

Phages specific for mycobacterial lipoarabinomannan help serodiagnosis of tuberculosis.

This study evaluated the possibility to use six phages specific to the Mycobacterium tuberculosis lipoarabinomannan (LAM) as tools for tubercular serodiagnosis. We analysed sera samples from 30 subjects with active tuberculosis (TB+), 30 with latent tubercular infection (LTBI) and 60 healthy subjects as controls (K). Our data indicated a good antibody response of the TB+ and LTBI patients against the phage Ri(7)17; the optical density (OD) values obtained from sera patients was statistically significant when compared to the control samples. Our results confirm that phage display technology might be useful to develop new tools for diagnosis of tuberculosis.

Levels of different cytokines in women and men with asymptomatic genital infection caused by Chlamydia.

INTRODUCTION: Immune response to genital Chlamydia trachomatis infection is involved in both immunity and pathology. The cytokine profile during infection has been implicated in the disease outcome, either resolution or severe sequelae. METHODOLOGY: In total, 3900 patients were analyzed for presence of genital infections caused by Chlamydia using molecular assays. Interleukins (IL) IL-10, IL-17, IL-6, IL-2 and chemokine IP-10 were estimated by ELISA in urine, cervical swabs and semen samples. Statistical analysis was performed using the T student test. RESULTS: A total of 47 out of 3900 samples (1.2%) were found to be positive for Chlamydia trachomatis based on the Real Time (RT) PCR results. Statistical analysis revealed that the differences between Chlamydia trachomatis positive and negative samples regarding levels of cytokines were not significant. CONCLUSIONS: Our results demonstrated that no significant difference in cytokine concentrations exists in Chlamydia trachomatis infected patients when compared to healthy controls. In further study, we aim to test on a greater number of positive samples a greater number of cytokines involved in the immune response to Chlamydia trachomatis infections.

Enhancement of antimicrobial activity of pump inhibitors associating drugs.

INTRODUCTION: with the continuous emergence of pathogenic resistance to conventional drugs through efflux pumps, increasing efforts are directed toward discovering efflux inhibitory molecules. METHODOLOGY: in this study three P-glycoprotein (P13CP, P22CP, P34CP) efflux-inhibitors (EIs), belonging to the series of phenoxymethylquinoxalines capable to restore/potentiate the antiproliferative activity of doxorubicin and vincristine against human tumor cell lines and different antibiotics against clinical isolates, were investigated on 10 clinical strains of Candida and 12 clinical and ATCC strains of Gram positive and Gram-negative bacteria. RESULTS: MFC values of FLC were reduced in all Candida strains by the P22CP and P34CP inhibitors, and in 5/10 fungal strains by the P13CP inhibitor. CONCLUSION: novel antibiotics with new modes of action are urgently required to suppress the rise of MDR bacteria. An alternative approach would be to identify molecules that can interfere with the process of efflux.

Design, synthesis and antitubercular activity of 4-alkoxy-triazoloquinolones able to inhibit the M. tuberculosis DNA gyrase.

A number of new F-triazolequinolones (FTQs) and alkoxy-triazolequinolones (ATQs) were designed, synthesized and evaluated for their activity against Mycobacterium tuberculosis H37Rv. Five out of 21 compounds exhibited interesting minimum inhibitory concentration (MIC) values (6.6-57.9 µM), ATQs generally being more potent than FTQs. Two ATQs, 21a and 30a, were endowed with the best anti-Mtb potency (MIC = 6.9 and 6.6 µM, respectively), and were not cytotoxic in a Vero cell line. Tested for activity against M. tuberculosis DNA gyrase in a DNA supercoiling activity assay, 21a and 30a showed IC50 values (27-28 µM) comparable to that of ciprofloxacin (10.6 µM). 21a was next selected for screening against several Mtb strains obtained from clinical isolates, including multi-drug-resistant (MDR) variants. Importantly, this compound was effective in all cases, with very promising MIC values (4 µM) in the case of some isoniazid/rifampicin-resistant Mtb strains. Finally, computer-based simulations revealed that the binding mode of 21a in the Mtb gyrase cleavage core complexed with DNA and the relevant network of intermolecular interactions are utterly similar to those described for ciprofloxacin, yielding a molecular rationale for the comparable anti-mycobacterial and DNA gyrase inhibition activity of this quinolone.

Performance of QuantiFERON-TB testing in a tuberculosis outbreak at a primary school.

This study compared the effectiveness of the QuantiFERON-TB Gold (QFT) assay with the Mantoux tuberculin skin test to detect Mycobacterium tuberculosis infection in 29 children during a school outbreak of tuberculosis. Of the 21 children with M. tuberculosis infection, 11 had a radiograph suggestive of the infection. The QFT assay was positive in all 21 of the children, and the Mantoux test was negative at first testing in 2 children (1 of whom was the sentinel case). The findings demonstrate that the QFT test is extremely useful in accurately identifying infected and uninfected children, permitting rapid intervention.

Heparin binding haemagglutin as potential diagnostic marker of Mycobacterium bovis.

In this study, we characterized the humoral responses in cattle of Sardinia. The animals were divided into three groups: 1) 28 cattle infected with Mycobacterium bovis; 2) 48 cattle from herds in which foci of infection was notified; 3) 50 cattle from herds that were TB-free. Levels of IgG antibody were measured against the following antigens of M. tuberculosis: Heparin-Binding-Haemagglutin (HBHA), Ag85B, PPE44, and PE_PGRS33 to investigate their potential to diagnose TB in animals. Our results indicated that HBHA is a potential candidate for the development of a serological assay for rapid diagnosis of cattle infected with M. bovis.

Identification of mycobacterial infections in wild boars in Northern Sardinia, Italy.

During a six-month period a region of Northern Sardinia was monitored to check the presence of mycobacterial infections in wild boars. Forty-eight serum and 229 biopsy samples were collected from different animals and examined by both traditional diagnostic techniques (culture, bacterioscopic and molecular tests) and enzyme-linked immunosorbent assay (ELISA). The latter was used to determine the antibody response against both methylated and nonmethylated Heparin-Binding Haemagglutinin (HBHA) protein. Nine mycobacterial strains were isolated: three M. avium ssp. paratuberculosis (Map), three M. avium, one M. interjectum and two M. scrofulaceum strains. By PCR, only one animal was positive for M. bovis, whereas 10 animals were positive for Map. Out of the 48 sera tested, 19 showed a good humoral response to methylated HBHA and 17 to nonmethylated HBHA. Our data provide new information on the prevalence of mycobacterial infection among wild boars in Northern Sardinia and suggest that a more effective program should be developed to monitor mycobacterial infections in the wild animal population.

QuantiFERON TB Gold: a new method for latent tuberculosis infection.

QuantiFERON-TB Gold obtained approval in 2003 by the Food and Drug Administration as a valid tool for the diagnosis of latent tuberculosis. In this report, we evaluated its potential use in the immunological diagnosis of Mycobacterium tuberculosis infections in different groups of subjects. Our data indicate that QuantiFERON-TB Gold is specific for identifying subjects who have come into contact with M. tuberculosis and its use alongside traditional diagnostic techniques may be an important instrument for controlling tuberculosis.

PE_PGRS proteins are differentially expressed by Mycobacterium tuberculosis in host tissues.

Characterization of PE_PGRS gene expression will help define the role of this protein family in the biology of Mycobacterium tuberculosis. In this report, quantitative real-time RT-PCR (QRT-PCR) was implemented to assess expression of three PE_PGRS genes (rv0746, rv1651c and rv1818c) under different experimental conditions. The three PE_PGRS genes showed a similar expression profile in axenic cultures, with a significant up-regulation occurring at late log and early stationary phases. rv1651c gene expression increased following intracellular growth in bone marrow-derived macrophages but not in type-II human pneumocytes, while rv0746 was induced in both in vitro systems. Following the infection of mice with M. tuberculosis, expression levels of rv1651c and rv0746 normalized to ftsZ and 16S rRNA were highest in the spleen tissue during the chronic stages of murine tuberculosis, with a >20- and >30-fold up-regulation, respectively. Levels of expression remained lower in the lung over the same time period. Expression of the rv1818c gene did not change significantly under different experimental conditions tested. The results of this study indicate that M. tuberculosis can differentially regulate expression of PE_PGRS genes and that genes such as rv0746 and rv1651c are significantly induced while M. tuberculosis persists in host cells and tissues.

In vitro efficacy of Linezolid on clinical strains of Mycobacterium tuberculosis and other mycobacteria.

Linezolid, an oxazolidinone that acts by inhibiting protein synthesis, was evaluated in strains of tuberculosis and non-tubercular mycobacteria resistant to one or more drugs isolated in northern Sardinia. The in vitro activity of Linezolid (Pfizer) was assessed on different isolates of Mycobacterium spp. from clinical samples by the Proportional Method. Linezolid demonstrated an excellent activity against the 24 strains of M. tuberculosis and against M. gordonae, M. marinum, M. aurum, M. phlei, and M. avium, with MIC values ranging from 0.5 to 2 microg/ml. Linezolid can be used in combination with the standard antitubercular medications, or as an effective therapeutic alternative in infections caused by M. tuberculosis or by other species of non-tubercular mycobacteria.

Tuberculosis in Sardinia: An investigation into the relationship between natives and immigrants.

OBJECTIVE/BACKGROUND: Tuberculosis (TB) has had a recrudescence in the last few decades in Italy as a result of many factors, among which migration from countries where TB is endemic is one of them. In Sardinia, a major island of Italy, there was no knowledge of the mechanisms of transmission of TB in the immigrant subpopulation and the impact it may have on the native subpopulation and on the community as a whole. Therefore, a molecular epidemiological study was carried out to get a clearer picture of the number and genetic features of Mycobacterium tuberculosis strains isolated from immigrants and from natives in Sardinia. METHODS: Two groups of clinical isolates of M. tuberculosis, one collected from immigrants and the other one from Sardinians, were analyzed in this study. The genotyping was executed through the variable number tandem repeat-mycobacterial interspersed repetitive units technique and a first-line antimycobacterial drug-susceptibility test was also carried out. RESULTS: Thirty-six clinical isolates from immigrants and 25 from Sardinians were analyzed. Variable number tandem repeat-mycobacterial interspersed repetitive units technique showed that all of them belonged to different strains and there was a quite high allelic diversity among them. Moreover, data collected allowed the finding of, with a good approximation, the phylogenetic relations among the strains isolated and the best-known phylogenetic groups. CONCLUSION: The study pointed out that since every strain is different, there was no TB transmission in any of the subpopulations and between immigrants and natives. This showed that the presence of immigrants was not a risk factor for contracting TB in the community.

Could inducible protein-10 and heparin-binding hemagglutinin improve the detection of Mycobacterium tuberculosis-infected subjects in a country with low incidence of tuberculosis?

BACKGROUND: This study aimed to evaluate inducible protein-10 (IP-10) as a biomarker besides interferon-gamma (IFN-γ) to improve the identification of active tuberculosis (TB) and latent tubercular infection (LTBI) in a country with a low incidence of TB. METHODS: Whole blood from Mycobacterium tuberculosis-infected subjects was stimulated with region-of-difference-1 (RD1)-specific peptides and with heparin-binding hemagglutinin (HBHA) to determine the release of IP-10 and IFN-γ. RESULTS: No statistically significant difference was observed between positive rates of IP-10 and IFN-γ after RD1-specific peptide stimulation in the TB and LTBI groups; a different response was detected in QuantiFERON TB-gold test-negative (QFT-) subjects. A significantly different proportion of positive responses was observed between IP-10 and IFN-γ following HBHA stimulation in the TB group and in the QFT- group but not in the LTBI group. CONCLUSIONS: The IP-10 test seemed to identify false-negative QFT results in some subjects with a positive IFN-γ/IP-10/HBHA pattern.