Conventional and real-time PCR targeting 16S ribosomal RNA for the detection of Mycobacterium tuberculosis complex.

SETTING: Conventional diagnostic methods for tuberculosis (TB) have limited sensitivity and specificity or are time-consuming. OBJECTIVE: 16S rDNA and 16S rRNA of Mycobacterium tuberculosis complex (MTC) were used as targets to develop sensitive and specific polymerase chain reactions (PCRs) to improve the diagnosis of MTC. DESIGN: We developed conventional and real-time PCRs targeting 16S rDNA and rRNA of MTC. RESULTS: PCRs targeting 16S rRNA had a 10-100 times lower limit of detection for M. tuberculosis than PCRs targeting 16S rDNA. The sensitivities of the 16S rDNA PCR, 16S rRNA reverse transcription PCR (RT-PCR), 16S rDNA real-time PCR and 16S rRNA real-time RT-PCR for sputum specimens were respectively 92%, 94.6%, 96% and 100%. Real-time PCR showed no cross-reactivity, but conventional PCR had cross-reactivity to M. avium, M. gastri and M. nonchromogenicum. CONCLUSION: PCRs targeting the 16S rRNA of MTC were more sensitive than those targeting 16S rDNA; 16S rRNA real-time RT-PCR showed the highest sensitivity and specificity for MTC.

Identification of Mycobacterium species in FFPE granulomatous lymphadenitis tissue using REBA Myco-ID®.

SETTING: Tuberculous lymphadenitis is the most common form of lymphadenopathy; its main histopathological finding is granulomatous inflammation. OBJECTIVE: A reverse blot hybridisation assay, REBA Myco-ID®, was applied to formalin-fixed paraffin-embedded (FFPE) tissue showing granulomatous lymphadenitis to define the causative agents. DESIGN: A total of 119 granulomatous lymphadenitis cases observed between 2000 and 2010 were studied. All tissue samples were treated by haematoxylin and eosin and Ziehl-Neelsen stain. Mycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) were identified using the REBA Myco-ID assay, and resistance to rifampicin (RMP) and isoniazid (INH) was determined using REBA MTB-MDR®. RESULTS: Of the 119 cases, 113 (95%) were positive with the REBA Myco-ID assay, while 20 (16.8%) were positive on acid-fast bacilli smear. Of the 113 positive REBA Myco-ID cases, 110 (92.43%) were identified as M. tuberculosis, 2 (1.7%) as NTM, and 1 (0.8%) as co-infection with M. tuberculosis and M. chelonae. Only 1 (0.9%) of the 110 M. tuberculosis cases was identified as RMP-resistant. CONCLUSION: REBA Myco-ID is a highly sensitive and specific assay for detecting M. tuberculosis and NTM. M. tuberculosis is the main cause of granulomatous lymphadenitis.

Mtb32 is a promising tuberculosis antigen for DNA vaccination in pre- and post-exposure mouse models.

Identification of antigens that provide protective immunity via prophylactic and therapeutic vaccination against Mycobacterium tuberculosis is critical for the development of subunit vaccines for tuberculosis (TB). In this study, we performed a head-to-head comparison of seven well-known TB antigens delivered by DNA vaccine, and evaluated their respective immunogenicities and protective efficacies in pre- and post-exposure mouse models. All TB antigens were designed as a chimeric fusion with Flt3-L to enhance antigen-specific T-cell immunity upon vaccination. Prophylactic vaccination with the Flt3L (F)-Mtb32 DNA vaccine elicited significant protection in both the spleen and lungs against M. tuberculosis challenge, comparable to the Bacillus Calmette-Guerin vaccine. F-Ag85A and F-Mtb32 DNA vaccines, in combination with chemotherapy, reduced the bacterial burden to undetectable levels in the lungs of all mice infected with M. tuberculosis. These data collectively indicate that the F-Mtb32 DNA vaccine confers the most efficient protective immunity that suppresses bacterial growth in the active or latent status of M. tuberculosis.

Bacille Calmette Guérin (BCG) can induce Kawasaki disease-like features in programmed death-1 (PD-1) gene knockout mice.

OBJECTIVES: Various genetic variants of inhibitory immune signals have been suspected as feasible causes of Kawasaki disease (KD). We investigated the associative role of programmed death-1 (PD-1) gene in the pathogenesis of KD by injecting bacilli Calmette Guérin (BCG) to PD-1 gene knockout (PD-1KO) mice. METHODS: In order to induce KD-like clinical manifestations in young PD-1KO mice, intradermal injection of the bacilli Calmette Guérin (BCG) was performed twice on the abdominal skin with a 4-week interval. For defining the role of BCG, heat shock protein (HSP) 65 was challenged. In addition, Staphylococcus aureus was adopted as a microorganism that does not contain HSP65 structure. One month after the second injection, heart, liver, and kidneys were removed and examined. RESULTS: PD-1KO mice showed KD-like features including prolonged fever for more than 5 days, erythematous swelling on soles, tail skin desquamation, and gallbladder (GB) hydrops. Inflammatory cell aggregation and intimal proliferation in at least more than one coronary artery was found in all PD-1KO mice whereas scanty coronary lesion was found in wild type (WT) mice. When the PD-1KO mice were injected twice with HSP65, coronary arterial lesions similar to those seen after BCG injection were observed. Inflammatory reactions in other organs including hepatic arteries, renal arteries, and biliary arteries were also observed in PD-1KO mice. CONCLUSIONS: Our data suggest that PD-1 gene may be one of the genetic predispositions of KD and antigens containing HSP65 structure could be a triggering factor of KD by our animal model of KD.

Satisfaction with mammography in the National Cancer Screening Programme participants of age 40s in Korea.

The aim of this study was to evaluate satisfaction with the National Cancer Screening Programme of mammography in Korea and to examine the association between subscales of satisfaction and general satisfaction. We conducted a cross-sectional telephone survey for women who had obtained a National Cancer Screening Programme mammographic screening at general hospitals between May and October 2008. The present study included 2005 women in their forties. We performed multivariate linear regression using dependent variable as general satisfaction and independent variables as subscales of satisfaction, such as pre-screening information transfer, staff interpersonal skills, physical surroundings and results reporting. Participants were stratified according to the result of their mammogram as negative or positive. Mean score of satisfaction was above 2.5 of 4 for all subscales. Women who received positive results were less satisfied with all of subscale factors. Staff interpersonal skills were the most important factor that contributed to general satisfaction. Future efforts such as staff training programme of communication/attitude skills, ensuring privacy and explanation of possible discomfort of the screening would be needed.

Tissue-specific changes in mRNA expression of Abc and Slc transporters in murine pulmonary tuberculosis.

A pulmonary tuberculosis mouse model was used to assess the pharmacodynamic and pharmacokinetic characteristics of tuberculosis therapeutics. While membrane transporters play important roles in drug disposition and physiological homeostasis, their expressional changes and contribution have never been analysed in a tuberculosis animal model. The mRNA expression level of 20 Abc family transporters and 32 Slc family transporters in tuberculosis-infected mice were compared with those in naïve uninfected mice using real-time polymerase chain reaction (PCR). Mycobacterium tuberculosis infection induced many dramatic expression changes of families of both Abc transporters and Slc transporters at 4 and 8 weeks, as observed in the livers, kidneys, and intestines of test mice--and in a different mode, in the lungs and spleens as well. These changes were dependent on the tuberculosis progression with the tissue-specific manner, that is, in the lungs, the number of transporters of which the expression level changed due to M. tuberculosis infection had increased, and the magnitude of change also greater at 8 weeks, while in the spleen, the transcription of most transporters except Mrps had not changed or had recovered back to the same level of naïve transcription at 8 weeks. Understanding the expression changes of transporters will assist in setting up rational preclinical dosing plans through the ability to predict the pharmacokinetics of new anti-tuberculosis chemotherapeutics and, furthermore, will assist in the design of safer and more efficient drug regimens.

Protection of mice against Mycobacterium tuberculosis infection by immunization with aqueous fraction of Triton X-100-soluble cell wall proteins.

The aqueous fraction of Triton X-100-soluble proteins (TSP-Aq) of Mycobacterium tuberculosis cell wall was reported to stimulate T-cell responses in peripheral blood monocytes from tuberculosis (TB) patients and to induce Th1 cytokines, suggesting presence of protective antigens. In this study, therefore, we examined the protective efficacy of TSP-Aq against M. tuberculosis infection in a mouse model. C57BL/6 mice were immunized with TSP-Aq or culture filtrate proteins (CFP) mixed with incomplete Freund's adjuvant or with BCG followed by i.v. challenge with M. tuberculosis H37Rv. TSP-Aq induced strong interferon-gamma production by spleen cells, and mice immunized with TSP-Aq antigens gave a significant reduction in M. tuberculosis CFU counts by 1.17-1.32 log10 CFU in the lungs and 1.31-2.08 log10 CFU in the spleen from 6 to 28 weeks. The degree of protection offered by TSP-Aq was comparable to that of CFP and of the BCG vaccine. The results demonstrated that the TSP-Aq antigens confer a significant level of protection against the growth of the organism in the lungs and spleen in a mouse model of TB and indicate that TSP contains major protective antigens of M. tuberculosis.

Protective effect of DNA vaccine during chemotherapy on reactivation and reinfection of Mycobacterium tuberculosis.

Active disease of tuberculosis (TB) can be developed decades later by either a relapse of the initial infection (endogenous reactivation) or by an entrance of the secondary infection (exogenous reinfection), since the current chemotherapy cannot lead to complete elimination of tuberculosis. Although the immunotherapeutic approaches in conjunction with conventional chemotherapy were tried to prevent TB growth via boosting the immune system, their therapeutic effects are still controversial. Here, we found that TB DNA vaccination completely blocked tuberculosis reactivation and significantly prevented from the secondary infection when chemotherapy was combined simultaneously. In particular, double-gene DNA vaccine composed of Ag85A and PstS-3 genes could reduce bacteria growth better than single-gene DNA vaccine after a secondary reinfection, indicating a correlation between the breadth of Th1 IFN-gamma response and the efficacy of the protection from reinfection. Thus, we propose that multigene TB DNA immunotherapy including Ag85A and PstS-3 genes during the period of chemotherapy could benefit patients undergoing TB chemotherapy in prevention from exogenous reinfection as well as endogenous reactivation.

Therapeutic effect of DNA vaccines combined with chemotherapy in a latent infection model after aerosol infection of mice with Mycobacterium tuberculosis.

The prevention of Mycobacterium tuberculosis (M. tuberculosis) reactivation would greatly reduce the incidence of the disease, particularly among the elderly. Here, we evaluated the efficacy of DNA vaccine in combination with a conventional TB chemotherapy on the prevention of M. tuberculosis reactivation. Mice were treated with isoniazid and pyrazinamide for 3 months from 4 weeks after aerosol infection with M. tuberculosis H37Rv. During this period of chemotherapy, DNA immunization was performed three times monthly with an antigen 85A (Ag85A) DNA or an IL-12 mutant (IL-12N220L) DNA, which is known to lead to a reduction in the secretion of the p40 subunit, but not of a bioactive IL-12p70. The reactivation of M. tuberculosis was dramatically reduced in mice treated with either Ag85A DNA (P<0.01) or IL-12N220L DNA (P<0.05) in combination with chemotherapy, compared with control mice receiving only chemotherapy. Ag85A DNA vaccine showed higher IFN-gamma responses to Ag85A protein, but a lower response to culture filtrate than IL-12N220L DNA vaccine. In addition, Ag85A DNA vaccine prevented the reactivation of M. tuberculosis more efficiently than IL-12N220L DNA vaccine, indicating that Ag85A-specific IFN-gamma response might correlate with M. tuberculosis control. This study suggests that immunotherapy using Ag85A or IL-12N220L DNA vaccine combined with conventional chemotherapy might be effective clinically for the prevention of tuberculosis reactivation and may offer a more effective cure for humans than chemotherapy alone.