Development of real-time PCR for the differential detection and quantification of Ureaplasma urealyticum and Ureaplasma parvum.

Ureaplasma parvum and Ureaplasma urealyticum are recently recognized species of the genus Ureaplasma. In humans, Ureaplasma spp. can be found on mucosal surfaces, primarily in the respiratory and urogenital tracts. They have been implicated in various human diseases such as nongonococcal urethritis, intrauterine infections in association with adverse pregnancy outcome and fetal morbidity, and pneumonitis in immunocompromised hosts. We have developed two quantitative real-time PCR assays to differentially detect U. parvum and U. urealyticum. Based upon the sequence information of the urease gene (ureB), we designed two TaqMan primer and probe combinations specific for U. parvum and U. urealyticum, respectively. The assays did not react with nucleic acid preparations from 16 bacterial species commonly encountered in relevant clinical specimens, including seven urease-producing species. Each assay had a detection limit of approximately five copies per reaction of the respective gene target. The results suggest that these assays are both sensitive and specific for U. parvum and U. urealyticum. Further investigation of both assays using clinical specimens is appropriate.

Large-scale whole genome sequencing of M. tuberculosis provides insights into transmission in a high prevalence area.

To improve understanding of the factors influencing tuberculosis transmission and the role of pathogen variation, we sequenced all available specimens from patients diagnosed over 15 years in a whole district in Malawi. Mycobacterium tuberculosis lineages were assigned and transmission networks constructed, allowing ≤10 single nucleotide polymorphisms (SNPs) difference. We defined disease as due to recent infection if the network-determined source was within 5 years, and assessed transmissibility from forward transmissions resulting in disease. High-quality sequences were available for 1687 disease episodes (72% of all culture-positive episodes): 66% of patients linked to at least one other patient. The between-patient mutation rate was 0.26 SNPs/year (95% CI 0.21-0.31). We showed striking differences by lineage in the proportion of disease due to recent transmission and in transmissibility (highest for lineage-2 and lowest for lineage-1) that were not confounded by immigration, HIV status or drug resistance. Transmissions resulting in disease decreased markedly over time.

Detection of heat-stable enterotoxin genes among Australian Vibrio cholerae O1 strains.

DNA probes derived from the heat-stable enterotoxin gene of Vibrio cholerae non-O1 (stn), and the cholera toxin gene (ctx), were used to screen 199 strains of V. cholerae O1, which were isolated within Australia from 1977-1986. 13 environmental strains isolated from the riverine environment in Southeast Queensland in 1980 and 1981, hybridized with the stn and ctx DNA probes. The concentrated supernatant of 6 of these strains elicited fluid accumulation in the infant mouse assay both before and after heating at 100 degrees C for 5 min. Genetic relationships among the 13 stn+ strains were studied by a comparison of the rRNA-RFLPs (ribotyping) and by Southern blot analysis with a stn gene probe. The results indicate that there is a clonal relationship among the Australian stn+ strains and that there is an environmental reservoir of stn genes among Australian V. cholerae O1 isolates.

Human immunodeficiency virus increases the risk of tuberculosis due to recent re-infection in individuals with latent infection.

BACKGROUND: Human immunodeficiency virus associated tuberculosis (TB) disease can follow reactivation of latent Mycobacterium tuberculosis infection or recent (re-)infection with M. tuberculosis. If contemporary TB cases share identical M. tuberculosis strains (i.e., are 'clustered'), the episode is likely to have followed recent (re-)infection, irrespective of evidence of previous latent infection. METHODS: Individuals experiencing a first TB episode between 1996 and 2008 in Karonga District, Northern Malawi, were included if information on M. tuberculosis infection status (from tuberculin tests) before 1990 and a DNA fingerprint from the TB episode were available. We explored differences in proportion clustered by prior M. tuberculosis infection status and HIV status, adjusting for age, sex, bacille Calmette-Guérin scar status and time since tuberculin testing. RESULTS: Of 79 HIV-negative TB cases, those with previous M. tuberculosis infection were much less likely to be clustered than cases without prior infection (29% vs. 77%, adjusted OR = 0.15, 95%CI 0.04-0.59). Among 119 HIV-positive TB cases, clustering was similar in both groups (88% vs. 84%, adjusted OR = 1.85, 95%CI 0.41-8.29). DISCUSSION: HIV infection appears to increase the risk of TB following recent re-infection in patients with latent M. tuberculosis infection. Our results add to the mounting evidence that HIV-associated TB mainly follows recent M. tuberculosis infection.

An epidemiological study of Vibrio cholerae O1 in the Australian environment based on rRNA gene polymorphisms.

Since 1977, Vibrio cholerae O1 has been isolated from the Australian aquatic environment and periodically cholera cases have occurred following exposure to these environments. To study the relationships between clinical isolates and environmental isolates from rivers and aquatic life, widely distributed throughout the country, a wide range of molecular typing methods were employed. In this paper we report the analysis of the 180 Australian isolates (10 clinical and 170 environmental) using ribotyping. Seven ribotype patterns were observed among the Australian inaba isolates, 2 of which included all clinical inaba isolates and 84% environmental inaba isolates collected from 9 rivers and creeks in eastern Australia during an 8-year period. Isolates from epidemiologically related clinical cases, asymptomatic household contacts and sewage were indistinguishable. The ogawa isolates were more diverse, with 9 ribotypes observed among 24 isolates from 8 rivers during the same period. Ribotype patterns were not shared between the serotypes with the exception of one ogawa isolate which could be distinguished using PFGE. Ribotyping has been useful in confirming an association between epidemiologically related clinical isolates and the aquatic environment and the persistence of several clones of the O1 serovar in the Australian environment during an 8-year period.

Expression of the human heat shock protein 60 in thyroid, pancreatic, hepatic and adrenal autoimmunity.

Heat shock proteins (hsps) have been proposed to play a role in autoimmune disease. Their highly conserved nature and role as a common antigenic determinant throughout phylogeny has raised the possibility that they may act as cellular targets of an autoimmune response when their expression is altered in stressed tissue cells. Using an antibody to human hsp60 we have demonstrated a wide tissue distribution in normal tissues, including thymus, the degree of staining reflecting the content of mitochondria in the cells, consistent with the known mitochondrial location of this protein. Enhanced staining was also demonstrated in oncocytes (deeply eosinophilic cells which have greatly increased numbers of mitochondria) in both thyroid and adrenal autoimmune disease and also in unrelated conditions where oncocytic change was identified. No enhancement was demonstrated in target cells in organ specific autoimmune diseases where oncocytic change was not seen, for example islet cells in diabetes and bile duct cells in primary biliary cirrhosis. Thus, no alteration of hsp60 expression was demonstrated which was specific to the autoimmune diseases studied.