Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA.

Transrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (<100 bp) and low concentration of TB-specific fragments. We aimed to improve the diagnostic sensitivity of TB urine cfDNA by increasing recovery of short fragments during sample preparation. We developed a highly sensitive sequence-specific purification method that uses hybridization probes immobilized on magnetic beads to capture short TB cfDNA (50 bp) with 91.8% average efficiency. Combined with short-target PCR, the assay limit of detection was ≤5 copies of cfDNA in 10 ml urine. In a clinical cohort study in South Africa, our urine cfDNA assay had 83.7% sensitivity (95% CI: 71.0 to 91.5%) and 100% specificity (95% CI: 86.2 to 100%) for diagnosis of active pulmonary TB when using sputum Xpert MTB/RIF as the reference standard. The detected cfDNA concentration was 0.14 to 2,804 copies/ml (median 14.6 copies/ml) and was inversely correlated with CD4 count and days to culture positivity. Sensitivity was nonsignificantly higher in HIV-positive (88.2%) compared to HIV-negative patients (73.3%), and was not dependent on CD4 count. Sensitivity remained high in sputum smear-negative (76.0%) and urine lipoarabinomannan (LAM)-negative (76.5%) patients. With improved sample preparation, urine cfDNA is a viable biomarker for TB diagnosis. Our assay has the highest reported accuracy of any TB urine cfDNA test to date and has the potential to enable rapid non-sputum-based TB diagnosis across key underserved patient populations.

Mortality from adverse drug reactions in adult medical inpatients at four hospitals in South Africa: a cross-sectional survey.

AIMS: Fatal adverse drug reactions (ADRs) are important causes of death, but data from resource-limited settings are scarce. We determined the proportion of deaths in South African medical inpatients attributable to ADRs, and their preventability, stratified by human immunodeficiency virus (HIV) status. METHODS: We reviewed the folders of all patients who died over a 30 day period in the medical wards of four hospitals. We identified ADR-related deaths (deaths where an ADR was 'possible', 'probable' or 'certain' using WHO-UMC criteria and where the ADR contributed to death). We determined preventability according to previously published criteria. RESULTS: ADRs contributed to the death of 2.9% of medical admissions and 56 of 357 deaths (16%) were ADR-related. Tenofovir, rifampicin and co-trimoxazole were the most commonly implicated drugs. 43% of ADRs were considered preventable. The following factors were independently associated with ADR-related death: HIV-infected patients on antiretroviral therapy (adjusted odds ratio (aOR) 4.4, 95% confidence interval (CI) 1.6, 12), exposure to more than seven drugs (aOR 2.5, 95% CI 1.3, 4.8) and increasing comorbidity score (aOR 1.3, 95% CI 1.1, 1.7). CONCLUSIONS: In our setting, where HIV and tuberculosis are highly prevalent, fatal in-hospital ADRs were more common than reported in high income settings. Most deaths were attributed to drugs used in managing HIV and tuberculosis. A large proportion of the ADRs were preventable, highlighting the need to strengthen systems for health care worker training and support.

Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing.

BACKGROUND: Urine cell-free DNA (cfDNA) is an attractive target for diagnosing pulmonary Mycobacterium tuberculosis (MTB) infection, but has not been thoroughly characterized as a biomarker. METHODS: This study was performed to investigate the size and composition of urine cfDNA from tuberculosis (TB) patients with minimal bias using next-generation sequencing (NGS). A combination of DNA extraction and single-stranded sequence library preparation methods demonstrated to recover short, highly degraded cfDNA fragments was employed. Urine cfDNA from 10 HIV-positive patients with pulmonary TB and two MTB-negative controls was examined. RESULTS: MTB-derived cfDNA was identifiable by NGS from all MTB-positive patients and was absent from negative controls. MTB cfDNA was significantly shorter than human cfDNA, with median fragment lengths of ≤19-52 bp and 42-92 bp, respectively. MTB cfDNA abundance increased exponentially with decreased fragment length, having a peak fragment length of ≤19 bp in most samples. In addition, we identified a larger fraction of short human genomic cfDNA, ranging from 29 to 53 bp, than previously reported. Urine cfDNA fragments spanned the MTB genome with relative uniformity, but nucleic acids derived from multicopy elements were proportionately over-represented. CONCLUSIONS: TB urine cfDNA is a potentially powerful biomarker but is highly fragmented, necessitating special procedures to maximize its recovery and detection.

Diagnostic outcomes of bone marrow aspirate and trephine biopsies performed at a hospital in KwaZulu-Natal, South Africa.

BACKGROUND: Bone marrow aspiration and trephine biopsy (BMAT) are widely performed in adults to evaluate haematological and malignant conditions. However, the diagnostic yield from the procedure in unselected patients in the South African public sector has not previously been described. OBJECTIVES: We identified the main indications and most common diagnoses encountered for BMAT and described the demographic and blood profiles of patients, including HIV-positive patients, who had undergone the procedure at a tertiary hospital in KwaZulu-Natal. METHODS: We retrospectively reviewed laboratory data from January 2016 to December 2016 for all patients aged ≥ 13 years who underwent the procedure and stratified findings by demographic data. RESULTS: Among 120 BMAT biopsies studied, 80 (67%) cases were performed to evaluate suspected malignancy and a further 40 (33%) cases for non-malignant indications. The main indications for bone marrow examination were: cytopenias 38 (32%), lymphoma 35 (29%), leukaemia 21 (18%), and multiple myeloma 17 (14%). BMAT results revealed that 60 cases (50%) were malignant in origin, 30 cases (25%) were non-malignant and 30 cases (25%) were classified as normal. The common diagnoses were: leukaemia, 24 (20%); multiple myeloma, 16 (13%) and lymphoma, 13 (11%). Cases aged ≥ 50 years were more likely to have a malignant diagnosis (odds ratio: 5.8 (95% confidence interval: 2.2-17.1) p-value < 0.001). CONCLUSION: The diagnostic yield of BMAT was high, with significant abnormalities detected in three quarters of cases. Haematological malignancy was the more common diagnosis. Increasing age was associated with an increase in reporting of haematology malignancy.

Impact of chemokine C-C ligand 27, foreskin anatomy and sexually transmitted infections on HIV-1 target cell availability in adolescent South African males.

We compared outer and inner foreskin tissue from adolescent males undergoing medical male circumcision to better understand signals that increase HIV target cell availability in the foreskin. We measured chemokine gene expression and the impact of sexually transmitted infections (STIs) on the density and location of T and Langerhans cells. Chemokine C-C ligand 27 (CCL27) was expressed 6.94-fold higher in the inner foreskin when compared with the outer foreskin. We show that the density of CD4+CCR5+ cells/mm2 was higher in the epithelium of the inner foreskin, regardless of STI status, in parallel with higher CCL27 gene expression. In the presence of STIs, there were higher numbers of CD4+CCR5+ cells/mm2 cells in the sub-stratum of the outer and inner foreskin with concurrently higher number of CD207+ Langerhans cells (LC) in both tissues, with the latter cells being closer to the keratin surface of the outer FS in the presence of an STI. When we tested the ability of exogenous CCL27 to induce T-cell migration in foreskin tissue, CD4 + T cells were able to relocate to the inner foreskin epithelium in response. We provide novel insight into the impact CCL27 and STIs on immune and HIV-1 target cell changes in the foreskin.

Cost-effectiveness of urine-based tuberculosis screening in hospitalised patients with HIV in Africa: a microsimulation modelling study.

BACKGROUND: Testing urine improves the number of tuberculosis diagnoses made among patients in hospital with HIV. In conjunction with the two-country randomised Rapid Urine-based Screening for Tuberculosis to Reduce AIDS-related Mortality in Hospitalised Patients in Africa (STAMP) trial, we used a microsimulation model to estimate the effects on clinical outcomes and the cost-effectiveness of adding urine-based tuberculosis screening to sputum screening for hospitalised patients with HIV. METHODS: We compared two tuberculosis screening strategies used irrespective of symptoms among hospitalised patients with HIV in Malawi and South Africa: a GeneXpert assay (Cepheid, Sunnyvale, CA, USA) for Mycobacterium tuberculosis and rifampicin resistance (Xpert) in sputum samples (standard of care) versus sputum Xpert combined with a lateral flow assay for M tuberculosis lipoarabinomannan in urine (Determine TB-LAM Ag test, Abbott, Waltham, MA, USA [formerly Alere]; TB-LAM) and concentrated urine Xpert (intervention). A cohort of simulated patients was modelled using selected characteristics of participants, tuberculosis diagnostic yields, and use of hospital resources in the STAMP trial. We calibrated 2-month model outputs to the STAMP trial results and projected clinical and economic outcomes at 2 years, 5 years, and over a lifetime. We judged the intervention to be cost-effective if the incremental cost-effectiveness ratio (ICER) was less than US$750/year of life saved (YLS) in Malawi and $940/YLS in South Africa. A modified intervention of adding only TB-LAM to the standard of care was also evaluated. We did a budget impact analysis of countrywide implementation of the intervention. FINDINGS: The intervention increased life expectancy by 0·5-1·2 years and was cost-effective, with an ICER of $450/YLS in Malawi and $840/YLS in South Africa. The ICERs decreased over time. At lifetime horizon, the intervention remained cost-effective under nearly all modelled assumptions. The modified intervention was at least as cost-effective as the intervention (ICERs $420/YLS in Malawi and $810/YLS in South Africa). Over 5 years, the intervention would save around 51 000 years of life in Malawi and around 171 000 years of life in South Africa. Health-care expenditure for screened individuals was estimated to increase by $37 million (10·8%) and $261 million (2·8%), respectively. INTERPRETATION: Urine-based tuberculosis screening of all hospitalised patients with HIV could increase life expectancy and be cost-effective in resource-limited settings. Urine TB-LAM is especially attractive because of high incremental diagnostic yield and low additional cost compared with sputum Xpert, making a compelling case for expanding its use to all hospitalised patients with HIV in areas with high HIV burden and endemic tuberculosis. FUNDING: UK Medical Research Council, UK Department for International Development, Wellcome Trust, US National Institutes of Health, Royal College of Physicians, Massachusetts General Hospital.

Structure-based optimization of protein tyrosine phosphatase 1B inhibitors: from the active site to the second phosphotyrosine binding site.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin and leptin receptor pathways and thus an attractive therapeutic target for diabetes and obesity. Starting with a high micromolar lead compound, structure-based optimization of novel PTP1B inhibitors by extension of the molecule from the enzyme active site into the second phosphotyrosine binding site is described. Medicinal chemistry, guided by X-ray complex structure and molecular modeling, has yielded low nanomolar PTP1B inhibitors in an efficient manner. Compounds from this chemical series were found to be actively transported into hepatocytes. This active uptake into target tissues could be one of the possible avenues to overcome the poor membrane permeability of PTP1B inhibitors.

Adverse Drug Reactions Causing Admission to Medical Wards: A Cross-Sectional Survey at 4 Hospitals in South Africa.

Limited data exist on the burden of serious adverse drug reactions (ADRs) in sub-Saharan Africa, which has high HIV and tuberculosis prevalence. We determined the proportion of adult admissions attributable to ADRs at 4 hospitals in South Africa. We characterized drugs implicated in, risk factors for, and the preventability of ADR-related admissions.We prospectively followed patients admitted to 4 hospitals' medical wards over sequential 30-day periods in 2013 and identified suspected ADRs with the aid of a trigger tool. A multidisciplinary team performed causality, preventability, and severity assessment using published criteria. We categorized an admission as ADR-related if the ADR was the primary reason for admission.There were 1951 admissions involving 1904 patients: median age was 50 years (interquartile range 34-65), 1057 of 1904 (56%) were female, 559 of 1904 (29%) were HIV-infected, and 183 of 1904 (10%) were on antituberculosis therapy (ATT). There were 164 of 1951 (8.4%) ADR-related admissions. After adjustment for age and ATT, ADR-related admission was independently associated (P ≤ 0.02) with female sex (adjusted odds ratio [aOR] 1.51, 95% confidence interval [95% CI] 1.06-2.14), increasing drug count (aOR 1.14 per additional drug, 95% CI 1.09-1.20), increasing comorbidity score (aOR 1.23 per additional point, 95% CI 1.07-1.41), and use of antiretroviral therapy (ART) if HIV-infected (aOR 1.92 compared with HIV-negative/unknown, 95% CI 1.17-3.14). The most common ADRs were renal impairment, hypoglycemia, liver injury, and hemorrhage. Tenofovir disoproxil fumarate, insulin, rifampicin, and warfarin were most commonly implicated, respectively, in these 4 ADRs. ART, ATT, and/or co-trimoxazole were implicated in 56 of 164 (34%) ADR-related admissions. Seventy-three of 164 (45%) ADRs were assessed as preventable.In our survey, approximately 1 in 12 admissions was because of an ADR. The range of ADRs and implicated drugs reflect South Africa's high HIV and tuberculosis burden. Identification and management of these ADRs should be considered in HIV and tuberculosis care and treatment programs and should be emphasized in health care worker training programmes.

A highly facile and efficient one-step synthesis of N6-adenosine and N6-2'-deoxyadenosine derivatives.

[reaction: see text] A highly facile and efficient one-step synthesis of N6-adenosine and N6-2'-deoxyadenosine derivatives has been developed. Treatment of inosine or 2'-deoxyinosine, without protection of sugar hydroxyl groups, with alkyl or arylamines, in the presence of BOP and DIPEA in DMF, led to the formation of N6-adenosine and N6-2'-deoxyadenosine derivatives in good to excellent yields. Carcinogenic polyaromatic hydrocarbon (PAH) N6-2'-deoxyadenosine adduct 10 and a rare DNA constituent 11 were thus synthesized directly from 2'-deoxyinosine both in 98% yield.