Facilitators and barriers to adolescent participation in a TB clinical trial.

The inclusion of adolescents in TB drug trials is essential for the development of safe, child-friendly regimens for the prevention and treatment of TB. TB Trials Consortium Study 31/AIDS Clinical Trials Group A5349 (S31/A5349) enrolled adolescents as young as 12 years old. We assessed investigator and coordinator described facilitators and barriers to adolescent recruitment, enrollment, and retention.Interviews were conducted with six investigators from sites that enrolled adolescent participants and six investigators from non-enrolling sites. Additionally, two focus groups were conducted with study coordinators from enrolling sites and two focus groups with non-enrolling sites. Discussions were transcribed, analyzed, summarized, and summaries were reviewed by Community Research Advisors Group members and research group representatives for content validity.Investigators and coordinators attributed the successful enrollment of adolescents to the establishment and cultivation of external partnerships, flexibility to accommodate adolescents' schedules, staff engagement, recruitment from multiple locations, dedicated recruitment staff working onsite to access potential participants, creation of youth-friendly environments, and effective communications. Non-enrolling sites were mainly hindered by regulations. Suggestions for improvement in future trials focused on study planning and site preparations.Proactive partnerships and collaboration with institutions serving adolescents helped identify and reduce barriers to their inclusion in this trial..

Challenges associated with electronic and in-person directly observed therapy during a randomized trial.

BACKGROUND: Electronic directly observed therapy (eDOT) has been proposed as an alternative to traditional in-person DOT (ipDOT) for monitoring TB treatment adherence. Information about the comparative performance and implementation of eDOT is limited.METHODS: The frequency of challenges during DOT, challenge type, and effect on medication observation were documented by DOT method during a crossover, noninferiority randomized controlled trial. A logistic mixed-effects model that adjusted for the study design was used to estimate the percentage of successfully observed doses when challenges occurred.RESULTS: A total of 20,097 medication doses were scheduled for observation with either eDOT (15,405/20,097; 76.7%) or ipDOT (4,692/20,097; 23.3%) for 213 study participants. In total, one or more challenges occurred during 17.3% (2,672/15,405) of eDOT sessions and 15.6% (730/4,692) of ipDOT sessions. Among 4,374 documented challenges, 27.3% (n = 1,192) were characterized as technical, 65.9% (n = 2,881) were patient-related, and 6.9% (n = 301) were program-related. Estimated from the logistic model (n = 6,782 doses, 173 participants), the adjusted percentage of doses successfully observed during problematic sessions was 21.7% (95% CI 11.2-37.8) for eDOT and 4.2% (95% CI 1.1-14.7) for ipDOT.CONCLUSION: Compared to ipDOT, challenges were encountered in a slightly higher percentage of eDOT sessions but were more often resolved to enable successful dose observation during problematic sessions.

Adverse events among persons with TB using in-person vs. electronic directly observed therapy.

BACKGROUND: We evaluated patient safety within a randomized crossover trial comparing electronic directly observed therapy (eDOT) to in-person DOT (ipDOT) in persons undergoing TB treatment in New York City, NY, USA.METHODS: Participant symptoms, symptom severity, and clinical management were documented. We assessed adverse event reports (AERs) by DOT method during the two-period crossover. Using Cox proportional-hazards mixed-effects models, we estimated the adjusted hazard ratio (aHR) of participants reporting an adverse event (AE) vs. not reporting an AE.RESULTS: Of 211 participants, 57 (27.0%) reported AEs during the two-period crossover; of these, 54.4% (31/57) were reported while using eDOT vs. 45.6% (26/57) while using ipDOT. Controlling for study group and period, the aHR for eDOT vs. ipDOT was 0.98 (95% CI 0.49-1.93). Although statistically not significant, the wide confidence intervals suggest that a significant association cannot be entirely ruled out. Gastrointestinal symptoms were most frequently reported (42.1%, 24/57). AER types and severity did not differ significantly by DOT method. Days from symptom onset to medical attention was similar across DOT methods (median: 1.0 day, IQR 0.0-2.0). No participants switched DOT methods due to AERs or monitoring concerns.CONCLUSION: Further evaluation to ascertain whether AERs differ when patients use eDOT vs. ipDOT is warranted.

Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family.

Mammalian natural resistance-associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe(2+), Mn(2+), Zn(2+), and Cu(2+). Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) also encode an Nramp homologue (Mramp). RNA encoding Mramp induces approximately 20-fold increases in (65)Zn(2+) and (55)Fe(2+) uptake when injected into Xenopus laevis oocytes. Transport is dependent on acidic extracellular pH and is maximal between pH 5.5 and 6.5. Mramp-mediated (65)Zn(2+) and (55)Fe(2+) transport is abolished by an excess of Mn(2+) and Cu(2+), confirming that Mramp interacts with a broad range of divalent transition metal cations. Using semiquantitative reverse transcription PCR, we show that Mramp mRNA levels in M. tuberculosis are upregulated in response to increases in ambient Fe(2+) and Cu(2+) between <1 and 5 microM concentrations and that this upregulation occurs in parallel with mRNA for y39, a putative metal-transporting P-type ATPase. Using a quantitative ratiometric PCR technique, we demonstrate a fourfold decrease in Mramp/y39 mRNA ratios from organisms grown in 5-70 microM Cu(2+). M. bovis BCG cultured axenically and within THP-1 cells also expresses mRNA encoding Mramp. Mramp exemplifies a novel prokaryotic class of metal ion transporter. Within phagosomes, Mramp and Nramp1 may compete for the same divalent cations, with implications for intracellular survival of mycobacteria.

Granulocyte colony-stimulating factor-induced upregulation of Jak3 transcription during granulocytic differentiation is mediated by the cooperative action of Sp1 and Stat3.

We previously demonstrated that Jak3 is a primary response gene for G-CSF and ectopic overexpression of Jak3 can accelerate granulocytic differentiation of normal mouse bone marrow cells induced by G-CSF and GM-CSF. To gain insight into the regulation of G-CSF-induced transcription of Jak3, we constructed deletion and linker scanning mutants of the Jak3 promoter sequences and performed luciferase reporter assays in the murine myeloid cell line 32Dcl3, with and without G-CSF stimulation. These experiments showed that mutation of a -67 to -85 element, which contained a putative Sp1 binding site, or mutation of a -44 to -53 GAS element resulted in a marked reduction of Jak3 promoter activity. Electrophoretic mobility shift assays revealed that Sp1 and Stat3 present in nuclear lysates of 32Dcl3 cells stimulated with G-CSF can bind to the -67 to -85 element and -44 to -53 GAS element, respectively. In addition, cotransfection of a constitutively active mutant of Stat3 along with a Jak3 promoter/luciferase reporter resulted in enhanced Jak3 promoter activity. Together, these results demonstrate that activation of Jak3 transcription during G-CSF- induced granulocytic differentiation is mediated by the combined action of Sp1 and Stat3, a mechanism also shown to be important in IL-6-induced monocytic differentiation.

Evaluation of the Tuberculosis Infection Control Training Center, Tajikistan, 2014-2015.

SETTING: Training center on tuberculosis (TB) infection control (IC) for health care workers in the Central Asian Republics region. OBJECTIVE: To assess the effects of TB IC training courses conducted at the Tuberculosis Infection Control Training Center in Machiton, Tajikistan. DESIGN: Participants who participated in training (n = 89) during the first year of operation (April 2014-February 2015) were invited to participate in a post-training interview. RESULTS: Of the 89 participants, 84 (94%) completed the interview and expressed satisfaction with the training. Eighty (95%) participants reported meeting with workplace leadership to discuss the training. Of these, 69 (85%) reported discussing changes required to meet TB IC standards. Self-reported changes in TB IC practices at work facilities post training included the creation of TB IC committees, designation of a TB IC focal person, TB IC planning, policies to separate infectious patients in waiting rooms, provision of masks for infectious patients, development of cough etiquette policies, improved glove availability, hand hygiene programs, and TB IC posters in waiting rooms. CONCLUSIONS: Participant satisfaction and reported changes in TB IC activities illustrate the potential of these training courses to improve TB IC in the region. Future training courses may be tailored to specific audiences using a structured conceptual framework to impact administration, budgeting, and facilities management of TB IC practices.

Multidrug-resistant tuberculosis patients' views of interventions to reduce treatment loss to follow-up.

SETTING: Patients who initiated treatment for multidrug-resistant tuberculosis (MDR-TB) at 15 Programmatic Management of Drug-resistant Tuberculosis (PMDT) health facilities in the Philippines between July and December 2012. OBJECTIVES: To describe patients' views of current interventions, and suggest changes likely to reduce MDR-TB loss to follow-up. METHODS: In-depth interviews were conducted between April and July 2014 with MDR-TB patients who were undergoing treatment, had finished treatment at the time of the interview (controls), or had been lost to follow-up (LTFU). Responses were thematically analyzed. RESULTS: Interviews were conducted with 182 patients who were undergoing or had completed treatment and 91 LTFU patients. Views and suggestions could be thematically categorized as approaches to facilitate adherence or address barriers to adherence. The top themes were the need for transportation assistance or improvements to the current transportation assistance program, food assistance, and difficulties patients encountered related to their medications. These themes were addressed by respectively 63%, 60%, and 32% of the participants. CONCLUSIONS: A more patient-centered approach is needed to improve MDR-TB treatment adherence. Programs should strive to provide assistance that considers patient preferences, is adequate to cover actual costs or needs, and is delivered in a timely, uninterrupted manner.

Evaluating the strengths and weaknesses of tuberculosis educational activities for prisoners in Honduras.

SETTING: Prisons throughout Honduras. OBJECTIVE: To assess the strengths, weaknesses, and opportunities to improve non-standardized tuberculosis (TB) educational campaign materials utilized in prisons. DESIGN: A qualitative assessment was conducted. Prison staff completed a survey describing development and implementation of educational activities at each prison (n=25). Completed surveys with photographs or copies of educational materials were collected and cataloged. The content, literacy demand, illustrations, learning stimulation, and cultural appropriateness of cataloged items were assessed using an adapted version of the Suitability Assessment of Materials Instrument. Results from this assessment were examined in conjunction with information collected through focus groups conducted with prisoners and interviews with prison staff. RESULTS: Campaign strengths included the development and dissemination of print materials appropriate for individuals with low literacy levels. By actively engaging prisoners in the campaigns, materials often incorporated the language, values, and experiences encountered within prison culture. Content of print materials and presentations focused on correcting misperceptions and facilitating acceptance of TB control activities. Campaign weaknesses included the creation of illustrations that could perpetuate the stigma associated with TB and use of some materials in which the purpose was not apparent. CONCLUSIONS: The campaigns were generally suitable, and the evaluation allowed for their improvement.

Ecologic correlates of cancer mortality patterns in an industrialized urban population.

Cancer mortality data for the period 1968-80 were analyzed to examine whether the high cancer burden for the city of Philadelphia was "evenly" distributed spatially and, if not, whether this distribution could be associated with socioeconomic variables and air pollution. Areas with significantly higher-than-expected rates tended to cluster; lung cancer and non-lung cancers showed distinctive cluster patterns, which were evident only for males; female rates for any cancer or groups of cancers were almost uniformly distributed over the neighborhoods. Both the high lung cancer and the high non-lung cancer clusters were characterized by low socioeconomic status, but only the high lung cancer clusters exhibited high levels of air pollution. These neighborhood characteristics suggested that socioeconomic variables explain non-lung cancer mortality patterns; socioeconomic variables, together with air pollution, could be responsible for the observed lung cancer patterns. The uniform distribution of female lung cancer rates suggested that air pollution by itself cannot be a prominent factor in lung cancer mortality.

Multidrug-resistant tuberculosis patients lost to follow-up: self-reported readiness to restart treatment.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) patients lost to follow-up (LTFU) from Programmatic Management of Drug-resistant Tuberculosis facilities in the Philippines. OBJECTIVES: To gain insight into patients' readiness to return to treatment. METHODS: MDR-TB patients who initiated treatment and were categorized as LTFU were identified using TB registers, contacted, and asked to consent to an interview and medical record review. At the conclusion of the interview, patients' readiness to restart treatment was assessed and examined in relation to demographic, clinical, and interview data. Odds ratios were calculated. RESULTS: When asked if they would consider restarting MDR-TB treatment, 3% of the 89 participating patients reported that they had already restarted, 34% indicated that they wanted to restart, 33% had not considered restarting, 28% were undecided, and 2% had decided against restarting. Patients who wanted to restart treatment were more likely to report having borrowed money for TB-related expenses (OR 5.97, 95%CI 1.27-28.18), and were less likely to report being self-employed (OR 0.08, 95%CI 0.01-0.67), or perceive themselves at low or no risk for TB relapse (OR 0.30, 95%CI 0.08-0.96) than patients who did not indicate an interest in restarting treatment. CONCLUSIONS: Efforts to re-engage LTFU patients in care should consider financial barriers, knowledge gaps, and personal adherence challenges in patients.

Quantitation of Toxoplasma gondii DNA in a competitive nested polymerase chain reaction.

AIM: To quantify Toxoplasma gondii DNA using a specially constructed artificial template as competitor in a nested polymerase chain reaction (PCR). METHODS: The diagnostic assay was a nested PCR employing four primers that amplify part of the single copy gene for the P30 major surface antigen in T gondii. An artificial competitor containing the four primer binding sites was made first by creating a 216 bp deletion in the native 914 bp full length PCR product using restriction enzyme digestion, ligation of selected digestion fragments, and cloning the ligation product into an E coli plasmid vector for production. Competitive nested PCR using three different quantities of T gondii genomic DNA with four corresponding 10-fold dilutions of the artificial competitor was then performed, and the results visualised with agarose gel electrophoresis. A standard curve was drawn by plotting the T gondii to competitor ratio readings against log10 of the competitor readings. RESULTS: The band intensities on agarose gel showed quantitative amplification in competitive nested PCR. The amount of competitor required to achieve equal molar amounts of PCR products is calculated by reading off the value of the competitor where the T gondii to competitor ratio equals 1 on the standard curves. CONCLUSIONS: Competitive PCR is possible with a nested assay, and quantitative amplification is well preserved. The use of an artificial competitor containing the same primer binding sites as the target enables the absolute amount of T gondii DNA in unknown samples to be estimated. In addition, the competitor simultaneously serves as a control for detecting false negative results of failed reactions in individual assay runs.

Recognition of tissue cyst-specific antigens in reactivating toxoplasmosis.

Current serological tests do not discriminate between asymptomatic latent Toxoplasma gondii infection and reactivating toxoplasmosis, but timely therapeutic intervention before the development of symptoms would lead to major reductions in morbidity and permanent disability. This study developed a new enzyme-linked immunosorbent assay (ELISA) for antibody to T. gondii tissue cyst antigens and screened tissue cyst antigens by Western blot analysis to test the hypothesis that antibody recognition of T. gondii tissue cyst-derived antigen is a good indicator of reactivation disease. A total of 187 sera was tested by Sabin-Feldman dye test and tissue cyst ELISA, AIDS patients and patients with ocular disease were considered separately, as the exposure to parasite antigens may be different in these two groups. The dye test did not discriminate between immunocompetent and immunocompromised T. gondii seropositive patients or between active and quiescent toxoplasmosis. Tissue cyst ELISA demonstrated a raised specific antibody response in immunocompetent T. gondii seropositive patients and in quiescent HIV positive sera. These data support th view that the tissue cyst population is in a state of dynamic equilibrium. It is proposed that, in the immunocompetent host, tissue cyst development and rupture are under some degree of immune control, but that in the immunocompromised host this equilibrium is disturbed and reactivation disease results. Data from patients with reactivating ocular toxoplasmosis demonstrate that tissue cyst-specific antibody levels are not different in active and quiescent disease and indeed they are not significantly different from immunocompetent T. gondii seronegative sera. In the Western blot analysis of 57 HIV positive patient sera, eight antigens (65, 57, 49, 47, 36, 28, 26 and 18 kDa) were consistently recognised by one third or more of the sera tested, but no single antigen was diagnostic of quiescent or active toxoplasmosis. It is concluded that tissue cyst-derived antigens are not a reliable serological marker of reactivating toxoplasmosis.

Incidence and mortality of hypoalbuminemic patients in hospitalized veterans.

The purpose of this study was to determine the incidence of hypoalbuminemia (HAlb) in a hospitalized male veteran population. Computerized surveillance of all clinical serum albumin determinations was performed during a 60-day interval. Each patient with an albumin level of 3.4 g/100 ml or less was identified and classified as an A, B, C, or D patient, based on the lowest recorded albumin level. After classification, each patient was followed either until death or for a minimum of 60 days. From a population of 2060 hospitalized veterans, 509 were identified as having HAlb for an incidence of 24.7%. The 30-day mortality rate for these 509 patients was 24.6%. The highest mortality rate, 62.0%, was seen in 50 group D patients whose albumin levels fell to 2.0 or below. For all HAlb patient groups, a linear correlation could be demonstrated between the level of HAlb observed and the subsequent 30-day mortality rate. It is concluded that, in our hospitalized veteran population, HAlb is a common finding and is associated with an increased 30-day mortality rate.

Extraction of RNA from Intracellular Mycobacterium tuberculosis : Methods, Considerations, and Applications.

Pathogenicity in Mycobacterium tuberculosis may be thought of as a multifactorial process with both pathogen and host-response effector molecules contributing to the process of infection, leading either to immunopathology and disease or control of infection and long-term persistence. Little is known about this at a genetic level, but it is becoming recognized that bacterial virulence constitutes the correct temporal and spatial regulation of many genes that may be necessary for a particular phase in infection in response to specific environmental cues.

Total joint replacement: implication of cancelled operations for hospital costs and waiting list management.

OBJECTIVE: To identify aspects of provision of total joint replacements which could be improved. DESIGN: 10 month prospective study of hospital admissions and hospital costs for patients whose total joint replacement was cancelled. SETTING: Information and Waiting List Unit, Musgrave Park Regional Orthopaedic Service, Belfast. PATIENTS: 284 consecutive patients called for admission for total joint replacement. MAIN MEASURES: Costs of cancellation of operation after admission in terms of hotel and opportunity costs. RESULTS: 28(10%) planned operations were cancelled, 27 of which were avoidable cancellations. Five replacement patients were substituted on the theatre list, leaving 22(8%) of 232 operating theatre opportunities unused. Patients seen at assessment clinics within two months before admission had a significantly higher operation rate than those admitted from a routine waiting list (224/232(97%) v 32/52(62%), x2 = 58.6, df = 1; p < 0.005). Mean duration of hospital stay in 28 patients with cancelled operations was 1.92 days. Operating theatre opportunity costs were 73% of the total costs of cancelled total joint replacements. CONCLUSION: Patients on long waiting lists for surgery should be reassessed before admission to avoid wasting theatre opportunities, whose cost is the largest component of the total costs of cancelled operations.

Cytokine mRNA expression in Mycobacterium ulcerans-infected human skin and correlation with local inflammatory response.

Cytokine mRNA expression in biopsies of Mycobacterium ulcerans-infected human tissue was investigated using real-time PCR, and the findings were correlated with the clinical stages of disease and histopathologies. A broad range of cytokine mRNAs were detected in 16 early nodules and 28 late-stage ulcers, including those for the Th1 cytokines tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) and the Th2 cytokine interleukin 10 (IL-10). IFN-gamma was strongly expressed in both nodules and ulcers, suggesting that a Th1 response begins early in the disease. There was a significantly higher expression of IL-8 and other proinflammatory cytokines in results from 32 biopsies with neutrophilia than in those from 12 biopsies without acute inflammation. Ten tissue samples containing granulomas showed high mRNA expression for IFN-gamma, IL-1beta, IL-12p35, IL-12p40, IL-15, and TNF-alpha relative to 34 tissue samples without granulomas. These results suggest that the human immune response to M. ulcerans is similar to that seen with some other mycobacteria despite the presence of the toxin mycolactone in the tissues.