Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

Rapid Detection of Chlamydia trachomatis and Typing of the Lymphogranuloma venereum associated L-Serovars by TaqMan PCR

<p>Abstract</p> <p>Background</p> <p>Infection due to <it>Chlamydia trachomatis </it>is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men.</p> <p>Results</p> <p>The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (<it>omp-1</it>) -gene and a L-serovar-specific region of the polymorphic protein H (<it>pmp-H</it>) -gene for the detection of <it>Chlamydia trachomatis </it>is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2- and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific <it>omp-1</it>-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5–95,2%.</p> <p>In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for <it>C. trachomatis </it>we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male.</p> <p>Conclusion</p> <p>This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies.</p

Sodium Chloride Inhibits the Growth and Infective Capacity of the Amphibian Chytrid Fungus and Increases Host Survival Rates

The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently emerged pathogen that causes the infectious disease chytridiomycosis and has been implicated as a contributing factor in the global amphibian decline. Since its discovery, research has been focused on developing various methods of mitigating the impact of chytridiomycosis on amphibian hosts but little attention has been given to the role of antifungal agents that could be added to the host's environment. Sodium chloride is a known antifungal agent used routinely in the aquaculture industry and this study investigates its potential for use as a disease management tool in amphibian conservation. The effect of 0–5 ppt NaCl on the growth, motility and survival of the chytrid fungus when grown in culture media and its effect on the growth, infection load and survivorship of infected Peron's tree frogs (Litoria peronii) in captivity, was investigated. The results reveal that these concentrations do not negatively affect the survival of the host or the pathogen. However, concentrations greater than 3 ppt significantly reduced the growth and motility of the chytrid fungus compared to 0 ppt. Concentrations of 1–4 ppt NaCl were also associated with significantly lower host infection loads while infected hosts exposed to 3 and 4 ppt NaCl were found to have significantly higher survival rates. These results support the potential for NaCl to be used as an environmentally distributed antifungal agent for the prevention of chytridiomycosis in susceptible amphibian hosts. However, further research is required to identify any negative effects of salt exposure on both target and non-target organisms prior to implementation

Hepatitis C virus to hepatocellular carcinoma

Hepatitis C virus causes acute and chronic hepatitis and can lead to permanent liver damage and hepatocellular carcinoma (HCC) in a significant number of patients via oxidative stress, insulin resistance (IR), fibrosis, liver cirrhosis and HCV induced steatosis. HCV induced steatosis and oxidative stress causes steato-hepatitis and these pathways lead to liver injury or HCC in chronic HCV infection. Steatosis and oxidative stress crosstalk play an important role in liver damage in HCV infection. This Review illustrates viral and host factors which induce Oxidative stress, steatosis and leads toward HCC. It also expresses Molecular cascade which leads oxidative stress and steatosis to HCC

Strengthening Ethics: A Faith Perspective on Educational Research

This article discusses ethical guidelines from the viewpoint of the teachings of Islam and, although not ostensibly different, finds parallels in the manner in which ethics could be conceptualised in the context of research in education. It seeks an alignment between ethics from the perspective of being a professional engaged in educational research with a personal significance based on one’s belief, which takes a holistic notion of life. The aims of being ethical researchers seem to be shared in many ways: protection, honesty and integrity. However, there is an added dimension of being accountable to God. The article first locates research within the wider context of Higher Education. Thereafter, to facilitate an understanding of ethics, as viewed from the framework of the Qur’ān, it proceeds to consider the ethical requirements of a university in the West Midlands, England, as a case study to examine the congruence, if any, with some of the ethical teachings of Islam. Based on the preliminary research, this article argues that Muslim researchers in education should be cognisant that their responsibilities are wider. Firstly, they are stewards meaning that the care and well-being of others comes first. Stewardship is both a Biblical and Qur’ānic concept meaning to be guardians. Secondly, by conducting their research in ethical ways, it will mean that they will be performing good deeds. These are meritorious actions rewarded by God. It also clarifies to supervisors and ethics review committees the key principles which might inform the thinking of some researchers from the Muslim community and how their motivations would be ethical