Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing

Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.Fil: Engström, Anna. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Swedish Institute for Communicable Disease Control; SueciaFil: Morcillo, Nora Susana. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Imperiale, Belén Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Hoffner, Sven E.. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Swedish Institute for Communicable Disease Control; SueciaFil: Juréen, Pontus. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Swedish Institute for Communicable Disease Control; Sueci

How does comorbidity influence healthcare costs? A population-based cross-sectional study of depression, back pain and osteoarthritis

Objectives To analyse how comorbidity among patients with back pain, depression and osteoarthritis influences healthcare costs per patient. A special focus was made on the distribution of costs for primary healthcare compared with specialist care, hospital care and drugs. Design Population-based cross-sectional study. Setting The County of Östergötland, Sweden. Patients Data on diagnoses and healthcare costs for all 266 354 individuals between 20 and 75 years of age, who were residents of the County of Östergötland, Sweden, in the year 2006, were extracted from the local healthcare register and the national register of drug prescriptions. Main outcome measures The effects of comorbidity on healthcare costs were estimated as interactions in regression models that also included age, sex, number of other health conditions and education. Results The largest diagnosed group was back pain (11 178 patients) followed by depression (7412 patients) and osteoarthritis (5174 patients). The largest comorbidity subgroup was the combination of back pain and depression (772 patients), followed by the combination of back pain and osteoarthritis (527 patients) and the combination of depression and osteoarthritis (206 patients). For patients having both a depression diagnosis and a back pain diagnosis, there was a significant negative interaction effect on total healthcare costs. The average healthcare costs among patients with depression and back pain was SEK 11 806 lower for a patient with both diagnoses. In this comorbidity group, there were tendencies of a positive interaction for general practitioner visits and negative interactions for all other visits and hospital days. Small or no interactions at all were seen between depression diagnoses and osteoarthritis diagnoses. Conclusions A small increase in primary healthcare visits in comorbid back pain and depression patients was accompanied with a substantial reduction in total healthcare costs and in hospital costs. Our results can be of value in analysing the cost effects of comorbidity and how the coordination of primary and secondary care may have an impact on healthcare costs

A 2-pyridone-amide inhibitor targets the glucose metabolism pathway of Chlamydia trachomatis.

UnlabelledIn a screen for compounds that inhibit infectivity of the obligate intracellular pathogen Chlamydia trachomatis, we identified the 2-pyridone amide KSK120. A fluorescent KSK120 analogue was synthesized and observed to be associated with the C. trachomatis surface, suggesting that its target is bacterial. We isolated KSK120-resistant strains and determined that several resistance mutations are in genes that affect the uptake and use of glucose-6-phosphate (G-6P). Consistent with an effect on G-6P metabolism, treatment with KSK120 blocked glycogen accumulation. Interestingly, KSK120 did not affect Escherichia coli or the host cell. Thus, 2-pyridone amides may represent a class of drugs that can specifically inhibit C. trachomatis infection.ImportanceChlamydia trachomatis is a bacterial pathogen of humans that causes a common sexually transmitted disease as well as eye infections. It grows only inside cells of its host organism, within a parasitophorous vacuole termed the inclusion. Little is known, however, about what bacterial components and processes are important for C. trachomatis cellular infectivity. Here, by using a visual screen for compounds that affect bacterial distribution within the chlamydial inclusion, we identified the inhibitor KSK120. As hypothesized, the altered bacterial distribution induced by KSK120 correlated with a block in C. trachomatis infectivity. Our data suggest that the compound targets the glucose-6-phosphate (G-6P) metabolism pathway of C. trachomatis, supporting previous indications that G-6P metabolism is critical for C. trachomatis infectivity. Thus, KSK120 may be a useful tool to study chlamydial glucose metabolism and has the potential to be used in the treatment of C. trachomatis infections

A randomized controlled trial comparing intensive non-surgical treatment with bariatric surgery in adolescents aged 13-16 years (AMOS2): Rationale, study design, and patient recruitment.

BACKGROUND: Previous non-randomized studies show similar outcomes in adolescents and adults after bariatric surgery. We describe the study protocol, recruitment, and selected baseline data of patients in a randomized multi-center study, the Adolescent Morbid Obesity Surgery 2 (AMOS2). METHODS: Three clinics in Sweden collaborated in designing the study and recruitment of patients from August 1, 2014 to June 30, 2017. Patients were selected among adolescents 13-16 years of age attending third-level obesity care for at least one year. Patients were randomized 1:1 to bariatric surgery (predominantly Roux-en-Y gastric bypass) or intensive non-surgical treatment starting with an eight-week low-calorie-diet. RESULTS: Fifty adolescents (37 girls) were randomized, 25 (19 girls) to bariatric surgery. Mean age was 15.7 years (range 13.3-16.9), weight 122.6 kg (range 95-183.3), Body Mass Index (BMI) 42.6 kg/m2 (range 35.7-54.9) and BMI-SDS 3.45 (range 2.9-4.1). One patient had type 2 diabetes mellitus, and 12/45 (27%) had elevated liver enzymes. There were no significant differences between the groups. For the 39 eligible patients who were offered but declined inclusion, BMI was not different from included patients. However, patients who declined were younger, 15.2 years (p = 0.021). A sex difference was also noted with more of eligible girls, 37/53 (69.8%), than boys, 13/36 (36.1%), wanting to participate in the study (p = 0.002). CONCLUSIONS: This clinical trial, randomizing adolescents with severe obesity to bariatric surgery or intensive non-surgical treatment, aims at informing about whether it is beneficial to undergo bariatric surgery in early adolescence. It will also enlighten the outcome of comprehensive non-surgical treatment. The study was registered at www.clinicalTrials.gov number NCT02378259

The importance of comorbidity in analysing patient costs in Swedish primary care

BACKGROUND: The objective was to explore the usefulness of the morbidity risk adjustment system Adjusted Clinical Groups(® )(ACG), in comparison with age and gender, in explaining and estimating patient costs on an individual level in Swedish primary health care. Data were retrieved from two primary health care centres in southeastern Sweden. METHODS: A cross-sectional observational study. Data from electronic patient registers from the two centres were retrieved for 2001 and 2002, and patients were grouped into ACGs, expressing the individual combination of diagnoses and thus the comorbidity. Costs per patient were calculated for both years in both centres. Cost data from one centre were used to create ACG weights. These weights were then applied to patients at the other centre. Correlations between individual patient costs, age, gender and ACG weights were studied. Multiple linear regression analyses were performed in order to explain and estimate patient costs. RESULTS: The variation in individual patient costs was substantial within age groups as well as within ACG weight groups. About 37.7% of the individual patient costs could be explained by ACG weights, and age and gender added about 0.8%. The individual patient costs in 2001 estimated 22.0% of patient costs in 2002, whereas ACG weights estimated 14.3%. CONCLUSION: ACGs was an important factor in explaining and estimating individual patient costs in primary health care. Costs were explained to only a minor extent by age and gender. However, the usefulness of the ACG system appears to be sensitive to the accuracy of classification and coding of diagnoses by physicians

Immunization of mice with the nef gene from Human Immunodeficiency Virus type 1: Study of immunological memory and long-term toxicology

<p>Abstract</p> <p>Background</p> <p>The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Nef, is an attractive vaccine target because it is involved in viral pathogenesis, is expressed early in the viral life cycle and harbors many T and B cell epitopes. Several clinical trials include gene-based vaccines encoding this protein. However, Nef has been shown to transform certain cell types <it>in vitro</it>. Based on these findings we performed a long-term toxicity and immunogenicity study of Nef, encoded either by Modified Vaccinia virus Ankara or by plasmid DNA. BALB/c mice were primed twice with either DNA or MVA encoding Nef and received a homologous or heterologous boost ten months later. In the meantime, the Nef-specific immune responses were monitored and at the time of sacrifice an extensive toxicological evaluation was performed, where presence of tumors and other pathological changes were assessed.</p> <p>Results</p> <p>The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs.</p> <p>Both DNAnef and MVAnef immunized animals developed potent Nef-specific cellular responses that declined to undetectable levels over time, and could readily be boosted after almost one year. This is of particular interest since it shows that plasmid DNA vaccine can also be used as a potent late booster of primed immune responses. We observed qualitative differences between the T cell responses induced by the two different vectors: DNA-encoded nef induced long-lasting CD8⁺T cell memory responses, whereas MVA-encoded nef induced CD4⁺T cell memory responses. In terms of the humoral immune responses, we show that two injections of MVAnef induce significant anti-Nef titers, while repeated injections of DNAnef do not. A single boost with MVAnef could enhance the antibody response following DNAnef prime to the same level as that observed in animals immunized repeatedly with MVAnef. We also demonstrate the possibility to boost HIV-1 Nef-specific immune responses using the MVAnef construct despite the presence of potent anti-vector immunity.</p> <p>Conclusion</p> <p>This study shows that the nef gene vectored by MVA does not induce malignancies or other adverse effects in mice. Further, we show that when the nef gene is delivered by plasmid or by a viral vector, it elicits potent and long-lasting immune responses and that these responses can be directed towards a CD4⁺or a CD8⁺T cell response depending on the choice of vector.</p

A 1.8 ps Time-to-Digital Converter (TDC) Implemented in a 20 nm Field-Programmable Gate Array (FPGA) Using a Ones-Counter Encoding Scheme with Embedded Bin-Width Calibrations and Temperature Correction

This thesis investigates the use of field-programmable gate arrays (FPGAs) to implement a time-to-digital converter (TDC) with on-chip calibration and temperature correction.Using carry-chains on the Xilinx Kintex UltraScale architecture to create a tapped delay line (TDL) has previously been proven to give good time resolution.This project improves the resolution further by using a bit-counter to handle bubbles in the TDL without removing any taps.The bit counter also adds the possibility of using a wave-union approach previously dismissed as unusable on this architecture.The final implementation achieves an RMS resolution of 1.8 ps

A 1.8 ps Time-to-Digital Converter (TDC) Implemented in a 20 nm Field-Programmable Gate Array (FPGA) Using a Ones-Counter Encoding Scheme with Embedded Bin-Width Calibrations and Temperature Correction

This thesis investigates the use of field-programmable gate arrays (FPGAs) to implement a time-to-digital converter (TDC) with on-chip calibration and temperature correction.Using carry-chains on the Xilinx Kintex UltraScale architecture to create a tapped delay line (TDL) has previously been proven to give good time resolution.This project improves the resolution further by using a bit-counter to handle bubbles in the TDL without removing any taps.The bit counter also adds the possibility of using a wave-union approach previously dismissed as unusable on this architecture.The final implementation achieves an RMS resolution of 1.8 ps