Technical Aspects for the Evaluation of Circulating Nucleic Acids (CNAs): Circulating Tumor DNA (ctDNA) and Circulating MicroRNAs

Circulating nucleic acids (CNAs), for example, circulating tumor DNA (ctDNA) and circulating microRNA (miRNA), represent promising biomarkers in several diseases including cancer. They can be isolated from many body fluids, such as blood, saliva, and urine. Also ascites, cerebrospinal fluids, and pleural effusion may be considered as a source of CNAs, but with several and intrinsic limitations. Therefore, blood withdrawal represents one of the best sources for CNAs due to the very simple and minimally invasive way of sampling. Moreover, it can be repeated at different time points, giving the opportunity for a real-time monitoring of the disease

Young people’s perceptions of smartphone-enabled self-testing and online care for sexually transmitted infections: qualitative interview study

Background Control of sexually transmitted infections (STI) is a global public health priority. Despite the UK’s free, confidential sexual health clinical services, those at greatest risk of STIs, including young people, report barriers to use. These include: embarrassment regarding face-to-face consultations; the time-commitment needed to attend clinic; privacy concerns (e.g. being seen attending clinic); and issues related to confidentiality. A smartphone-enabled STI self-testing device, linked with online clinical care pathways for treatment, partner notification, and disease surveillance, is being developed by the eSTI2 consortium. It is intended to benefit public health, and could do so by increasing testing among populations which underutilise existing services and/or by enabling rapid provision of effective treatment. We explored its acceptability among potential users. Methods In-depth interviews were conducted in 2012 with 25 sexually-experienced 16–24 year olds, recruited from Further Education colleges in an urban, high STI prevalence area. Thematic analysis was undertaken. Results Nine females and 16 males participated. 21 self-defined as Black; three, mixed ethnicity; and one, Muslim/Asian. 22 reported experience of STI testing, two reported previous STI diagnoses, and all had owned smartphones. Participants expressed enthusiasm about the proposed service, and suggested that they and their peers would use it and test more often if it were available. Utilizing sexual healthcare was perceived to be easier and faster with STI self-testing and online clinical care, which facilitated concealment of STI testing from peers/family, and avoided embarrassing face-to-face consultations. Despite these perceived advantages to privacy, new privacy concerns arose regarding communications technology: principally the risk inherent in having evidence of STI testing or diagnosis visible or retrievable on their phone. Some concerns arose regarding the proposed self-test’s accuracy, related to self-operation and the technology’s novelty. Several expressed anxiety around the possibility of being diagnosed and treated without any contact with healthcare professionals. Conclusions Remote STI self-testing and online care appealed to these young people. It addressed barriers they associated with conventional STI services, thus may benefit public health through earlier detection and treatment. Our findings underpin development of online care pathways, as part of ongoing research to create this complex e-health intervention

Detection of multiple strains of Mycobacterium tuberculosis using MIRU-VNTR in patients with pulmonary tuberculosis in Kampala, Uganda

<p>Abstract</p> <p>Background</p> <p>Many studies using DNA fingerprinting to differentiate <it>Mycobacterium tuberculosis </it>(MTB) strains reveal single strains in cultures, suggesting that most disease is caused by infection with a single strain. However, recent studies using molecular epidemiological tools that amplify multiple targets have demonstrated simultaneous infection with multiple strains of MTB. We aimed to determine the prevalence of MTB multiple strain infections in Kampala, and the impact of these infections on clinical presentation of tuberculosis (TB) and response to treatment.</p> <p>Methods</p> <p>A total of 113 consecutive smear and culture positive patients who previously enrolled in a house-hold contact study were included in this study. To determine whether infection with multiple MTB strains has a clinical impact on the initial presentation of patients, retrospective patient data (baseline clinical, radiological and drug susceptibility profiles) was obtained. To determine presence of infections with multiple MTB strains, MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeats) -PCR was performed on genomic DNA extracted from MTB cultures of smear positive sputum samples at baseline, second and fifth months.</p> <p>Results</p> <p>Of 113 patients, eight (7.1%) had infection with multiple MTB strains, coupled with a high rate of HIV infection (37.5% versus 12.6%, <it>p </it>= 0.049). The remaining patients (105) were infected with single MTB strains. The proportions of patients with MTB smear positive cultures after two and five months of treatment were similar. There was no difference between the two groups for other variables.</p> <p>Conclusion</p> <p>Infection with multiple MTB strains occurs among patients with first episode of pulmonary tuberculosis in Kampala, in a setting with high TB incidence. Infection with multiple MTB strains had little impact on the clinical course for individual patients. This is the first MIRU-VNTR-based study from in an East African country.</p

Clinical Deterioration during Antitubercular Treatment at a District Hospital in South Africa: The Importance of Drug Resistance and AIDS Defining Illnesses

Background: Clinical deterioration on drug therapy for tuberculosis is a common cause of hospital admission in Africa. Potential causes for clinical deterioration in settings of high HIV-1 prevalence include drug resistant Mycobacterium tuberculosis (M.tb), co-morbid illnesses, poor adherence to therapy, tuberculosis associated-immune reconstitution inflammatory syndrome (TB-IRIS) and subtherapeutic antitubercular drug levels. It is important to derive a rapid diagnostic work-up to determine the cause of clinical deterioration as well as specific management to prevent further clinical deterioration and death. We undertook this study among tuberculosis (TB) patients referred to an adult district level hospital situated in a high HIV-1 prevalence setting to determine the frequency, reasons and outcome for such clinical deterioration. Method: A prospective observational study conducted during the first quarter of 2007. We defined clinical deterioration as clinical worsening or failure to stabilise after 14 or more days of antitubercular treatment, resulting in hospital referral. We collected data on tuberculosis diagnosis and treatment, HIV-1 status and antiretroviral treatment, and investigated reasons for clinical deterioration as well as outcome. Results: During this period, 352 TB patients met inclusion criteria; 296 were admitted to hospital accounting for 17% of total medical admissions (n = 1755). Eighty three percent of TB patients (291/352) were known to be HIV-1 co-infected with a median CD4 count of 89cells/mm3 (IQR 38-157). Mortality among TB patients admitted to hospital was 16% (n = 48). The median duration of hospital admission was 9.5 days (IQR 4-18), longer than routine in this setting (4 days). Among patients in whom HIV-1 status was known (n = 324), 72% of TB patients (n = 232) had an additional illness to tuberculosis; new AIDS defining illnesses (n = 80) were the most frequent additional illnesses (n = 208) in HIV-1 co-infected patients (n = 291). Rifampin-resistant M.tb (n = 41), TB-IRIS (n = 51) and drug resistant bacterial infections (n = 12) were found in 12%, 14% and 3.4% of the 352 cases, respectively. Interpretation: In our setting, new AIDS defining illnesses, drug resistant M.tb and other drug resistant bacteria are important reasons for clinical deterioration in HIV-1 co-infected patients receiving antitubercular treatment. HIV-1 coinfected patients may be at increased risk of acquiring nosocomial drug resistant pathogens because profound immune suppression results in co-morbid illnesses that require prolonged inpatient admissions. Routine infection control is essential and needs to be strengthened in our setting. Copyright: © 2009 Pepper et al

Genomic and proteomic analyses of Mycobacterium bovis BCG Mexico 1931 reveal a diverse immunogenic repertoire against tuberculosis infection

<p>Abstract</p> <p>Background</p> <p>Studies of <it>Mycobacterium bovis </it>BCG strains used in different countries and vaccination programs show clear variations in the genomes and immune protective properties of BCG strains. The aim of this study was to characterise the genomic and immune proteomic profile of the BCG 1931 strain used in Mexico.</p> <p>Results</p> <p>BCG Mexico 1931 has a circular chromosome of 4,350,386 bp with a G+C content and numbers of genes and pseudogenes similar to those of BCG Tokyo and BCG Pasteur. BCG Mexico 1931 lacks Region of Difference 1 (RD1), RD2 and N-RD18 and one copy of IS6110, indicating that BCG Mexico 1931 belongs to DU2 group IV within the BCG vaccine genealogy. In addition, this strain contains three new RDs, which are 53 (RDMex01), 655 (RDMex02) and 2,847 bp (REDMex03) long, and 55 single-nucleotide polymorphisms representing non-synonymous mutations compared to BCG Pasteur and BCG Tokyo. In a comparative proteomic analysis, the BCG Mexico 1931, Danish, Phipps and Tokyo strains showed 812, 794, 791 and 701 protein spots, respectively. The same analysis showed that BCG Mexico 1931 shares 62% of its protein spots with the BCG Danish strain, 61% with the BCG Phipps strain and only 48% with the BCG Tokyo strain. Thirty-nine reactive spots were detected in BCG Mexico 1931 using sera from subjects with active tuberculosis infections and positive tuberculin skin tests.</p> <p>Conclusions</p> <p>BCG Mexico 1931 has a smaller genome than the BCG Pasteur and BCG Tokyo strains. Two specific deletions in BCG Mexico 1931 are described (RDMex02 and RDMex03). The loss of RDMex02 (<it>fadD23</it>) is associated with enhanced macrophage binding and RDMex03 contains genes that may be involved in regulatory pathways. We also describe new antigenic proteins for the first time.</p

Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis, and Concurrently Affect the Pathogen's Growth

BACKGROUND:The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung. METHODOLOGY/PRINCIPAL FINDINGS:During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage. CONCLUSIONS/SIGNIFICANCE:While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide's binding to Esat6-as the latter is not an essential protein of M. tuberculosis-nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen