Multicenter matched-pair study comparing BACT/ALERT® MP reagent systems for the recovery of mycobacteria from specimens other than blood.

The BACT/ALERT® MP Reagent System is a broth culture medium for optimal detection and recovery of mycobacteria from clinical samples. The MP formulation was recently modified to improve detection and recovery times. A multicenter prospective matched pair study design was conducted to validate the performance of improved MP (MP-I) versus current MP (MP-C) bottles utilizing nonsterile and normally sterile samples, except blood, from patients suspected of having mycobacterial infections. A total of 1488 clinical samples were collected to obtain 212 mycobacteria samples by either or both MP culture bottles. MP-I and MP-C sensitivities were 86.6% and 81.4%, respectively, but the difference was not significant (P = 0.163) while specificities were 96.8% and 93.8%, respectively, and that difference was significant (P = 0.002). Overall recovery was 94.34% for MP-I and 88.68% for MP-C (recovery was 100% for both bottles with 52 seeded samples). Overall performance of MP-I was better than MP-C for sensitivity, specificity, and recovery.

Aneuploidy in dizygotic twin sheep detected using genome-wide single nucleotide polymorphism data from two commonly used commercial vendors.

Early detection of karyotype abnormalities, including aneuploidy, could aid producers in identifying animals which, for example, would not be suitable candidate parents. Genome-wide genetic marker data in the form of single nucleotide polymorphisms (SNPs) are now being routinely generated on animals. The objective of the present study was to describe the statistics that could be generated from the allele intensity values from such SNP data to diagnose karyotype abnormalities; of particular interest was whether detection of aneuploidy was possible with both commonly used genotyping platforms in agricultural species, namely the Applied BiosystemsTM AxiomTM and the Illumina platform. The hypothesis was tested using a case study of a set of dizygotic X-chromosome monosomy 53,X sheep twins. Genome-wide SNP data were available from the Illumina platform (11 082 autosomal and 191 X-chromosome SNPs) on 1848 male and 8954 female sheep and available from the AxiomTM platform (11 128 autosomal and 68 X-chromosome SNPs) on 383 female sheep. Genotype allele intensity values, either as their original raw values or transformed to logarithm intensity ratio (LRR), were used to accurately diagnose two dizygotic (i.e. fraternal) twin 53,X sheep, both of which received their single X chromosome from their sire. This is the first reported case of 53,X dizygotic twins in any species. Relative to the X-chromosome SNP genotype mean allele intensity values of normal females, the mean allele intensity value of SNP genotypes on the X chromosome of the two females monosomic for the X chromosome was 7.45 to 12.4 standard deviations less, and were easily detectable using either the AxiomTM or Illumina genotype platform; the next lowest mean allele intensity value of a female was 4.71 or 3.3 standard deviations less than the population mean depending on the platform used. Both 53,X females could also be detected based on the genotype LRR although this was more easily detectable when comparing the mean LRR of the X chromosome of each female to the mean LRR of their respective autosomes. On autopsy, the ovaries of the two sheep were small for their age and evidence of prior ovulation was not appreciated. In both sheep, the density of primordial follicles in the ovarian cortex was lower than normally found in ovine ovaries and primary follicle development was not observed. Mammary gland development was very limited. Results substantiate previous studies in other species that aneuploidy can be readily detected using SNP genotype allele intensity values generally already available, and the approach proposed in the present study was agnostic to genotype platform.

DNA copy-number loss on 1p36.1 harboring RUNX3 with promoter hypermethylation and associated loss of RUNX3 expression in liver fluke-associated intrahepatic cholangiocarcinoma.

Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene, localized on 1p36, involved in TGF-beta-Smads signaling. To assess its role in liver fluke-associated intrahepatic cholangiocarcinoma (ICC), the promoter methylation status was investigated in 53 ICCs by methylation-specific PCR, with determination of loss of 1p36.1 by microarray comparative genomic hybridization and RUNX3 protein expression by immunohistochemistry. Loss at 1p36.1 was found 41.5% of ICCs (22/53). In addition, DNA hypermethylation of the RUNX3 promoter was found in 49.1% (26/53) of cancers and 57.1% (4/7) of ICC cell lines. The protein was highly expressed in normal bile ducts but mostly decreased in ICCs, 67.9% (n= 36) being negative for immunohistochemical staining. Promoter hypermethylation of RUNX3 was associated with reversible decrease or absence of RUNX3 protein expression (p<0.001), but this was not found to differ with the ICC subtype. In contrast, loss of 1p36.1 demonstrated a significant link (p= 0.020). In conclusion, RUNX3 promoter hypermethylation and loss of 1p36.1 are causal mechanisms for loss of RUNX3 function in liver fluke-associated ICC carcinogenesis.

Isolation of mycobacteria by BACTEC 460 TB system from clinical specimens.

This article reports our experience with the BACTEC 460 TB system in the past five years and its performance characteristics and its advantages over the conventional LJ medium for mycobacterial culture. Clinical specimens (3597) from patients suspected to have tuberculosis were submitted for mycobacterial culture between May 2000 and August 2005 and were processed using the BACTEC 460 TB system. Pulmonary samples were 1568 while the extra pulmonary samples were 2029. BACTEC achieved detection of 681 (18.93%) M. tuberculosis cases (499- pulmonary, 182- extrapulmonary) with a recovery time shorter by 13.2 days compared to conventional method, while 577 (84.7%) were non-tuberculosis mycobacteria. Automated systems can have a great impact and thrust on an early diagnosis of tuberculosis allowing an early and appropriate management of the patient and thereby a better disease outcome.

Determination of mycobacterial phylogeny on the basis of immunological relatedness of superoxide dismutases.

Sixteen strains of cultivable mycobacteria were grown in Sauton's medium, and Mycobacterium leprae was purified from armadillo liver. Cell extracts were prepared from log-phase growths of each of the cultivable mycobacterial strains. Superoxide dismutase (SOD) enzyme was purified from all cultivable mycobacterial strains included in the study, and antibodies against purified SOD enzyme were raised in rabbits. Immunological distances (ImDs) between these anti-SOD antibodies and SOD antigens were determined by a previously described immunoprecipitation method and by a recently developed enzyme-linked immunosorbent assay (ELISA) technique. The reciprocal ImDs among mycobacterial strains were constant, reproducible and consistent by these two methods. An evolutionary tree was constructed on the basis of estimated ImDs. Except for M. duvalii and M. terrae, slowly and rapidly growing mycobacterial species appeared to be separately grouped by this analysis. Rapid growers clustered into a group which is near that of some slow-growing mycobacteria. M. avium falls almost in the middle of the evolutionary tree and the position of M. leprae was found to be between those of M. avium and M. bovis BCG. Measurement of immunological relatedness of SODs provides an alternative system with which to study the taxonomical relatedness among mycobacteria.

Development of a SOD ELISA to determine the immunological relatedness among mycobacteria.

This study reports on the standardization of an enzyme-linked immunosorbent assay (ELISA) system for the measurement of immunological distances (ImDs) of the superoxide dismutase (SOD) molecule among the cultivable mycobacteria, namely, Mycobacterium vaccae, M. phlei, M. smegmatis, M. avium, M. scrofulaceum, M. tuberculosis H37Ra, M. tuberculosis H37Rv, and M. bovis BCG, and M. leprae. SODs from cultivable mycobacteria were purified, antibodies were raised against these molecules, and ImDs between these anti-SOD antibodies and antigen (SODs) were determined by an immunoprecipitation technique standardized earlier and by the ELISA technique developed in this study. The ELISA system developed in this study showed higher sensitivity and consistent and reproducible ImDs among various mycobacteria, including pathogens such as M. tuberculosis, M. leprae and M. avium. These values were comparable with the values derived by the immunoprecipitation technique. Our ELISA technique appears to be a sensitive and rapidly reproducible method with the additional advantage of the stability of reagents, and holds promise in the taxonomy as well as in the development of diagnostics for leprosy and other mycobacterial infections.

Treatment of bacilliferous BL/LL cases with combined chemotherapy and immunotherapy.

Thirty-six, untreated borderline lepromatous/lepromatous (BL/LL) leprosy patients with an initial bacterial index (BI) of 4+ to 6+ were serially allocated to three treatment groups. Group I patients received a slightly modified WHO regimen (rifampin once a month, clofazimine and dapsone daily) and BCG intradermally (i.d.) (0.1 mg/per dose). Group II patients were administered the same MDT and Mycobacterium w (2 x 10(8)) killed bacilli/dose i.d., and Group III received the same MDT with 0.1 ml of distilled water i.d. Vaccination was repeated every 6 months. Biopsies were taken from the local site of vaccination and from a distant site, i.e., the back. The progress was monitored periodically by clinical, histopathological and bacterial (BI, mouse foot pad, ATP) parameters. Twenty-five patients had completed a follow up of more than 2 years. These included: 7 in Group I, 10 in Group II, and 8 in Group III. One patient of the MDT + BCG group who was progressing well dropped out after 28 months. In cases on combined chemotherapy and immunotherapy, no viable bacilli were demonstrable by mouse foot pad and ATP measurement after 6 months (at 12 months or afterward). However, in come of the control cases on MDT alone, viable bacilli could be detected even up to 18 months (by mouse foot pad) and 2 years (by ATP estimation). With 36 months of treatment, the mean BI decreased from 4.64+ to 1.66+ in the group on MDT alone (controls), 4.9+ to 0.08+ in the MDT + BCG group, and 4.75+ to 0 in the MDT+Mycobacterium w group. Compared with the MDT and MDT + BCG groups, the fall in the BI was significantly more in the MDT + Mycobacterium w group at 12, 18, and 24 months. While all of the cases in the Mycobacterium w groups became smear negative by 36 months, it took 42 months for all of the BCG group to achieve negativity. Immunotherapy appears to have a significant effect on the killing and clearance of bacilli and should be considered as an adjunct to chemotherapy, especially in bacilliferous lepromatous cases.

Correlation between inhibitory effect of quinolones and mycolic acid metabolism in mycobacteria.

Mycolic acids are important components having a significant role in maintaining the rigidity of mycobacterial cell wall. They could also be the barrier for penetration of certain drugs into the bacterial cell. A novel in vitro model system was established for assessing the effect of Ciproflaxacin on mycolic acid metabolism in pathogenic mycobacteria M. Kansasii (which has similar mycolic acid pattern to that from M. leprae) and the effect of norfloxacin in M. intracellulare. These test mycobacteria were exposed in their midlogarithmic phase of growth to 0.5, 1, 2, 3, 4, 5 and 6 micrograms ml of ciprofloxacin and norfloxacin respectively for 1, 2 and 24 hours. Ciprofloxacin completely inhibited the synthesis of mycolates in M. kansasii at 3, 4 and 5 micrograms/ml; whereas norfloxacin exhibited its maximum inhibitory action on mycolic acids in M. intracellulare at 6 micrograms/ml for all the durations of exposure. Inhibition of mycolates directly correlated with bacterial viability which was estimated by colony forming units. The effect of quinolones on mycolic acid metabolism appears to be direct and not secondary to DNA gyrase. The results obtained from this study and our previous findings show that mycolic acid metabolism is affected by various groups of drugs, whose primary sites of activity may be different. The findings of the present study may have significant therapeutic implications in leprosy and other mycobacterial diseases.

Rapid characterization of Mycobacterium fortuitum-chelonei complex by restriction fragment length polymorphism of ribosomal RNA genes.

Using labelled, gamma-32P rRNA of mycobacteria as a probe restriction fragment length polymorphism (RFLP) of rRNA genes of strains belonging to the Mycobacterium fortuitum-chelonei complex was analysed. Each DNA sample was cleaved with EcoRI restriction endonuclease, the fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose membrane. Fragments of DNA containing rRNA genes were identified by hybridization with gamma-32P-labelled rRNA. Patterns were found to be species specific and both the species were distinguishable from each other. Results indicate that this approach can be used for rapid genomic characterization of the Mycobacterium fortuitum-chelonei complex.

Application of ATP assay for in vitro drug screening testing against human derived M. leprae.

In this study, the ATP content of M. leprae exposed to various antimicrobial agents has been measured to evaluate its usefulness in drug sensitivity screening. Purified M. leprae suspensions from human biopsies have been incubated at 30 degrees C in a modified Dubos medium in the presence of different concentrations of various drugs viz., Rifampicin, Ethionamide, Ethambutol, Cycloserine, Dapsone, Clofazimine, Erythromycin and Tetracycline. ATP levels were estimated at 0, 7 days, 14 days of incubation by the procedures modified and standardised at this laboratory. ATP decay was accelerated by ethionamide, rifampicin, clofazimine, dapsone, erythromycin and to a lesser extent by cycloserine, whereas ethambutol and tetracycline did not have any significant effect. The rate of decay depended on the concentrations of these drugs. ATP assay promises to be a useful system for in vitro drug sensitivity screening against M. leprae isolated from patients.