PURPOSE: PCR is widely used for rapidly and accurately detecting Mycobacterium Species. The purpose of this study was to assess the diagnostic performance of three real-time PCR kits and evaluate the concordance with two older PCR methods. MATERIALS AND METHODS: Using 128 samples, the five PCR methods were assessed, including an in-house PCR protocol, the COBAS Amplicor MTB, the COBAS TaqMan MTB, the AdvanSure TB/NTM real-time PCR, and the Real-Q M. tuberculosis kit. The discrepant results were further examined by DNA sequencing and using the AdvanSure Mycobacteria Genotyping Chip for complete analysis. RESULTS: For Mycobacterium tuberculosis (MTB) detection, all five kits showed 100% matching results (positive; N = 11 and negative; N = 80). In non-tuberculous mycobacterium (NTM) discrimination, the AdvanSure yielded two true-positive outcomes from M. intracellulare and one false positive outcome, while the Real-Q resulted in one true-positive outcome and one false negative outcome for each case and another false negative result using the provided DNA samples. CONCLUSION: Real-time PCR, yielded results that were comparable to those of the older PCR methods for detecting MTB. However, there were disagreements among the applied kits in regard to the sample test results for detecting NTM. Therefore, we recommend that additional confirmatory measures such as DNA sequencing should be implemented in such cases, and further research with using a larger numbers of samples is warranted to improve the detection of NTM.
DNA, Bacterial/genetics ; Humans ; Mycobacterium/*genetics ; Mycobacterium Infections/*diagnosis/microbiology ; Mycobacterium avium Complex/genetics ; Mycobacterium avium-intracellulare Infection/diagnosis ; Mycobacterium tuberculosis/genetics ; Polymerase Chain Reaction/*standards ; Reagent Kits, Diagnostic/*standards ; Tuberculosis/diagnosis

OBJECTIVEMycobacterium avium (M. avium) and Mycobacterium intracellulare (M. intracellulare) are the major causative agents of nontuberculous mycobacteria (NTM)-related pulmonary infections. However, little is known about the differences in drug susceptibility profiles between these two species.

METHODSA total of 393 NTM isolates were collected from Shanghai Pulmonary Disease Hospital. Sequencing of partial genes was performed to identify the strains at species level. The minimum inhibitory concentration (MIC) was used to evaluate the drug susceptibility against 20 antimicrobial agents. Variable number of tandem repeat (VNTR) typing was conducted to genotype these two species.

RESULTSA total of 173 (44.0%) M. avium complex (MAC) isolates were identified, including 41 (10.4%) M. avium isolates and 132 (33.6%) M. intracellulare isolates. Clarithromycin and amikacin were the two most effective agents against MAC isolates. The Hunter-Gaston Discriminatory Index (HGDI) values for VNTR typing of M. avium and M. intracellulare isolates were 0.993 and 0.995, respectively. Levofloxacin resistance was more common among the unclustered strains than among the clustered strains of M. intracellulare.

CONCLUSIONM. intracellulare was the most common NTM species in China. Clarithromycin and amikacin had high antimicrobial activities against MAC. VNTR typing of MAC isolates revealed a high discriminatory power. Levofloxacin resistance was associated with unclustered strains of M. intracellulare.

Anti-Bacterial Agents ; pharmacology ; Drug Resistance, Bacterial ; Genotype ; Humans ; Mycobacterium avium Complex ; drug effects ; genetics ; Mycobacterium avium-intracellulare Infection ; epidemiology ; microbiology

IS901 RFLP analysis of 36 Mycobacterium avium subsp. avium (MAA) isolates from 15 pheasants (Phasianus colchicus) and two goshawks (Accipiter gentilis) from four pheasant farms was performed. Using this method, six different IS901 RFLP types (E, F, G, M, Q, and V) were identified. The distribution of IS901 RFLP profiles was tightly linked to individual flocks. Matching IS901 RFLP profiles observed in the present study indicate MAA transmission between pheasants and goshawks in the same locality. In two flocks, different pheasants within a flock as well as in various organs of five individual pheasants were found to have two distinct IS901 RFLP profiles.
Animals ; Bone Marrow/microbiology ; *Galliformes ; Intestines/microbiology ; Liver/microbiology ; Mycobacterium avium/*genetics ; *Polymorphism, Genetic ; *Polymorphism, Restriction Fragment Length ; Poultry Diseases/*microbiology ; Spleen/microbiology ; Tuberculosis, Avian/*microbiology

Isolated bone marrow infection by nontuberculous mycobacteria (NTM) is extremely rare. Recently, we encountered a case of bone marrow Mycobacterium avium complex (MAC) infection, which presented as a fever of unknown origin shortly after starting continuous ambulatory peritoneal dialysis (CAPD). The patient was diagnosed with MAC infection on the basis of PCR-restriction fragment length polymorphism analysis and sequencing of DNA obtained from bone marrow specimens. Although this was a case of severe MAC infection, there was no evidence of infection of other organs. End-stage renal disease (ESRD) patients undergoing dialysis can be considered immunodeficient; therefore, when these patients present with fever of unknown origin, opportunistic infections such as NTM infection should be considered in the differential diagnosis.
Aged ; Anti-Bacterial Agents/therapeutic use ; Bacterial Proteins/genetics ; Bone Marrow/microbiology/pathology ; Diagnosis, Differential ; Female ; HIV Infections/diagnosis ; Humans ; Kidney Failure, Chronic/therapy ; *Mycobacterium avium Complex/genetics/isolation &purification ; Mycobacterium avium-intracellulare Infection/*diagnosis/drug therapy/microbiology ; *Peritoneal Dialysis, Continuous Ambulatory ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA

PCR is a highly accurate technique for confirming the presence of Mycobacterium avium subsp. paratuberculosis (Map) in broth culture. In this study, a simple, efficient, and low-cost method of harvesting DNA from Map cultured in liquid medium was developed. The proposed protocol (Universidad Austral de Chile [UACH]) was evaluated by comparing its performance to that of two traditional techniques (a QIAamp DNA Stool Mini Kit and cethyltrimethylammonium bromide [CTAB] method). The results were statistically assessed by agreement analysis for which differences in the number of cycles to positive (CP) were compared by Student's t-test for paired samples and regression analysis. Twelve out of 104 fecal pools cultured were positive. The final PCR results for 11 samples analyzed with the QIAamp and UACH methods or ones examined with the QIAamp and CTAB methods were in agreement. Complete (100%) agreement was observed between data from the CTAB and UACH methods. CP values for the UACH and CTAB techniques were not significantly different, while the UACH method yielded significantly lower CP values compared to the QIAamp kit. The proposed extraction method combines reliability and efficiency with simplicity and lower cost.
Animals ; Bacteriological Techniques/economics/*veterinary ; Cattle ; Cattle Diseases/diagnosis/*microbiology ; DNA, Bacterial/chemistry/genetics ; Female ; Mycobacterium avium subsp. paratuberculosis/*genetics ; Paratuberculosis/diagnosis/*microbiology ; Real-Time Polymerase Chain Reaction/veterinary ; Reproducibility of Results

The presence of galectin-3 was immunohistochemically quantified in bovine intestines infected with paratuberculosis (Johne's disease) to determine whether galectin-3 was involved in the formation of granulation tissue associated with the disease. Mycobacterium avium subsp. paratuberculosis infection was histochemically confirmed using Ziehl-Neelsen staining and molecularly diagnosed through rpoB DNA sequencing. Galectin-3 was detected in the majority of inflammatory cells, possibly macrophages, in the granulomatous lesions within affected tissues, including the ileum. These findings suggest that galectin-3 is associated with the formation of chronic granulation tissues in bovine paratuberculosis, probably through cell adhesion and anti-apoptosis mechanisms.
Animals ; Cattle ; Cattle Diseases/*pathology ; Chronic Disease ; Galectin 3/*metabolism ; Immunohistochemistry ; Intestine, Small/microbiology/*pathology ; Mycobacterium avium subsp. paratuberculosis/growth & development/isolation & purification ; Paratuberculosis/*pathology ; RNA Polymerase II/genetics

In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and anti-inflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4+/+ BMDCs was not observed in TLR4-/- BMDCs. Furthermore, FAP induced DC-mediated CD8+ T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway.
*Adhesins, Bacterial/genetics/metabolism ; Animals ; CD8-Positive T-Lymphocytes/metabolism ; *Cancer Vaccines/therapeutic use ; Cell Proliferation ; Cytokines/metabolism ; Dendritic Cells/*cytology ; Disease Models, Animal ; Gene Expression Regulation ; Glycogen Synthase Kinase 3/metabolism ; Humans ; Mice ; Mice, Inbred C57BL ; Mycobacterium avium/genetics/metabolism ; Paratuberculosis/metabolism ; Protein Binding ; Signal Transduction ; T-Lymphocytes, Cytotoxic/metabolism ; *Thymoma/genetics/metabolism ; *Toll-Like Receptor 4/agonists/genetics/metabolism