Doing No Harm? Adverse Events in a Nation-Wide Cohort of Patients with Multidrug-Resistant Tuberculosis in Nigeria

Background

Adverse events (AEs) of second line anti-tuberculosis drugs (SLDs) are relatively well documented. However, the actual burden has rarely been described in detail in programmatic settings. We investigated the occurrence of these events in the national cohort of multidrug-resistant tuberculosis (MDR-TB) patients in Nigeria.

Method

This was a retrospective, observational cohort study, using pharmacovigilance data systematically collected at all MDR-TB treatment centers in Nigeria. Characteristics of AEs during the intensive phase treatment were documented, and risk factors for development of AEs were assessed.

Results

Four hundred and sixty patients were included in the analysis: 62% were male; median age was 33 years [Interquartile Range (IQR):28–42] and median weight was 51 kg (IQR: 45–59). Two hundred and three (44%) patients experienced AEs; four died of conditions associated with SLD AEs. Gastro-intestinal (n = 100), neurological (n = 75), ototoxic (n = 72) and psychiatric (n = 60) AEs were the most commonly reported, whereas ototoxic and psychiatric AEs were the most debilitating. Majority of AEs developed after 1–2 months of therapy, and resolved in less than a month after treatment. Some treatment centers were twice as likely to report AEs compared with others, highlighting significant inconsistencies in reporting at different treatment centers. Patients with a higher body weight had an increased risk of experiencing AEs. No differences were observed in risk of AEs between HIV-infected and uninfected patients. Similarly, age was not significantly associated with AEs.

Conclusion

Patients in the Nigerian MDR-TB cohort experienced a wide range of AEs, some of which were disabling and fatal. Early identification and prompt management as well as standardized reporting of AEs at all levels of healthcare, including the community is urgently needed. Safer regimens for drug-resistant TB with the shortest duration are advocated.     

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative,Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing^TM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.     

Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

Introduction

Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity.

Methods

Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood.

Results

The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood.

Conclusion

Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.     

The Multi-Drug Resistant Tuberculosis Diagnosis and Treatment Cascade in Bangladesh

Objectives

To determine, in areas supported by BRAC, Bangladesh i) the pre-diagnosis and pre-treatment attrition among presumptive and confirmed Multi-Drug Resistant Tuberculosis (MDR-TB) patients and ii) factors associated with attrition.

Methods

This was a retrospective cohort study involving record review. Presumptive MDR-TB patients from peripheral microscopy centres serving 60% of the total population of Bangladesh were included in the study. Attrition and turnaround time for MDR-TB diagnosis by Xpert MTB/RIF and treatment initiation were calculated between July 2012 and June 2014.

Results

Of 836 presumptive MDR-TB patients referred from 398 peripheral microscopy centres, 161 MDR-TB patients were diagnosed. The number of diagnosed MDR-TB patients was less than country estimates of MDR-TB patients (2000 cases) during the study period. Among those referred, pre-diagnosis and pre-treatment attrition was 17% and 21% respectively. Median turnaround time for MDR-TB testing, result receipt and treatment initiation was four, zero and five days respectively. Farmers (RR=2.3, p=0.01) and daily wage laborers (RR=2.1, p=0.04) had twice the risk of having pre-diagnosis attrition. Poor record-keeping and unreliable upkeep of presumptive MDR-TB patient databases were identified as challenges at the peripheral microscopy centres.

Conclusion

There was a low proportion of pre-diagnosis and pre-treatment attrition in patients with presumptive and confirmed MDR-TB under programmatic conditions. However, the recording and reporting system did not detect all presumptive MDR-TB patients, highlighting the need to improve the system in order to prevent morbidity, mortality and transmission of MDR-TB in the community.     

Rural health centres, communities and malaria case detection in Zambia using mobile telephones: a means to detect potential reservoirs of infection in unstable transmission conditions

Background

Zoonotic malaria caused by Plasmodium knowlesi is an important, but newly recognized, human pathogen. For the first time, post-mortem findings from a fatal case of knowlesi malaria are reported here.

Case presentation

A formerly healthy 40 year-old male became symptomatic 10 days after spending time in the jungle of North Borneo. Four days later, he presented to hospital in a state of collapse and died within two hours. He was hyponatraemic and had elevated blood urea, potassium, lactate dehydrogenase and amino transferase values; he was also thrombocytopenic and eosinophilic. Dengue haemorrhagic shock was suspected and a post-mortem examination performed. Investigations for dengue virus were negative. Blood for malaria parasites indicated hyperparasitaemia and single species P. knowlesi infection was confirmed by nested-PCR. Macroscopic pathology of the brain and endocardium showed multiple petechial haemorrhages, the liver and spleen were enlarged and lungs had features consistent with ARDS. Microscopic pathology showed sequestration of pigmented parasitized red blood cells in the vessels of the cerebrum, cerebellum, heart and kidney without evidence of chronic inflammatory reaction in the brain or any other organ examined. Brain sections were negative for intracellular adhesion molecule-1. The spleen and liver had abundant pigment containing macrophages and parasitized red blood cells. The kidney had evidence of acute tubular necrosis and endothelial cells in heart sections were prominent.

Conclusions

The overall picture in this case was one of systemic malaria infection that fit the WHO classification for severe malaria. Post-mortem findings in this case were unexpectedly similar to those that define fatal falciparum malaria, including cerebral pathology. There were important differences including the absence of coma despite petechial haemorrhages and parasite sequestration in the brain. These results suggest that further study of knowlesi malaria will aid the interpretation of, often conflicting, information on malaria pathophysiology in humans.     

Recovery of antioxidant phenolics from white vinification solid by-products employing water/ethanol mixtures

Solid wastes from white vinification, including grape peels, seeds and stems, were used as raw material for the recovery of antioxidant polyphenols. Extractions were performed using non-toxic media composed of water/ethanol mixtures and hydrochloric, acetic or tartaric acid. Recovery efficiency was assessed by monitoring the antioxidant potency of extracts and several indices related to their polyphenolic composition, including total polyphenol, total flavonoid, total flavanol and condensed tannin (proanthocyanidin) content. Among the by-products tested, seeds were shown to contain exceptional amounts of total polyphenols (13.76 g per 100g dry weight), followed by stems (7.47 g per 100g dry weight) and peels (0.97 g per 100g dry weight). Extracts with the highest antioxidant activity from all by-products were obtained with 57% ethanol. Acidification of this medium with 0.1% HCl improved polyphenol recovery and antiradical activity for stem extracts, but it was unfavourable for seed extraction.     

Combining immunological and molecular data to assess phylogenetic relations of some Greek Podarcis species

Most recent molecular studies revealed the phylogeny of Greek Podarcis species, which for years remained elusive, due to discordant data produced from various chromosomal, complement fixation and protein studies. In this report, we analyzed cellular immune responses of spleen-derived lymphocytes from six allopatric Podarcis species encountered in Greece, by assessing two-way mixed lymphocyte reaction (MLR)-induced proliferation. On the basis of stimulation indices (S.I.) as determined from cultures set up from xenogeneic splenocytes coincubated in pairs, we generated a phylogenetic tree, fully consistent with the phylogenetic relationships of Podarcis as determined by parallel analyses based on partial mitochondrial (mt) DNA sequences. Although the exact mechanisms triggering lymphocyte responses in lizard two-way xenogeneic MLR are not fully understood, our results show the potential use of cell-mediated immune responses as an additional approach to mtDNA analysis, for species delimitation within specific lizard taxa.     

Evolutionary stability of sex chromosomes in snakes

Amniote vertebrates possess various mechanisms of sex determination, but their variability is not equally distributed. The large evolutionary stability of sex chromosomes in viviparous mammals and birds was believed to be connected with their endothermy. However, some ectotherm lineages seem to be comparably conserved in sex determination, but previously there was a lack of molecular evidence to confirm this. Here, we document a stability of sex chromosomes in advanced snakes based on the testing of Z-specificity of genes using quantitative PCR (qPCR) across 37 snake species (our qPCR technique is suitable for molecular sexing in potentially all advanced snakes). We discovered that at least part of sex chromosomes is homologous across all families of caenophidian snakes (Acrochordidae, Xenodermatidae, Pareatidae, Viperidae, Homalopsidae, Colubridae, Elapidae and Lamprophiidae). The emergence of differentiated sex chromosomes can be dated back to about 60 Ma and preceded the extensive diversification of advanced snakes, the group with more than 3000 species. The Z-specific genes of caenophidian snakes are (pseudo)autosomal in the members of the snake families Pythonidae, Xenopeltidae, Boidae, Erycidae and Sanziniidae, as well as in outgroups with differentiated sex chromosomes such as monitor lizards, iguanas and chameleons. Along with iguanas, advanced snakes are therefore another example of ectothermic amniotes with a long-term stability of sex chromosomes comparable with endotherms.     

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.