Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

PDXK mutations cause polyneuropathy responsive to pyridoxal 5'-phosphate supplementation.

OBJECTIVE: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. METHODS: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. RESULTS: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. INTERPRETATION: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels. ANN NEUROL 2019;86:225-240

Der Berner Patienten- und Therapeutenstundenbogen 2000

Theoretischer Hintergrund: Kontinuierliche Qualitätssicherung psychotherapeutischer Behandlung ist Grundlage für die adaptive Indikation und Aufrechterhaltung der Prozessqualität. Dazu werden Messinstrumente gefordert, die zentrale Aspekte des Psychotherapieprozesses möglichst umfassend und dennoch zeitökonomisch aus Therapeuten- und Patientensicht abbilden (Grawe, 1998). Fragestellung: Entspricht der Berner Therapeuten- und Patientenstundenbogen 2000 (TSTB/PSTB) zentralen messtheoretischen Anforderungen? Methode: Anhand von 429 ambulant behandelten Patienten wird die Faktorenstruktur in verschiedenen Settings überprüft sowie Fragen zur Reliabilität und Validität der Bögen beantwortet. Ergebnisse: Die konfirmatorischen Faktoranalysen bestätigen die theoriegeleiteten Skalen der Vorgängerversion über verschiedene Settings und Therapiephasen hinweg (TSTB: Therapiebeziehung, Offenheit, Therapiefortschritte, interaktionelle Schwierigkeit, Problembewältigung, Bezug zur realen Lebenssituation, motivationale Klärung, Ressourcenaktivierung, Problemaktualisierung, Anstrengungsbereitschaft, interaktionelle Perspektive; PSTB: Therapiebeziehung, Selbstwerterfahrungen, Bewältigungserfahrungen, Klärungserfahrungen, Therapiefortschritte, Aufgehobensein, Direktivität/Kontrollerfahrungen, Problemaktualisierung). Die Reliabilitäten der Mehr-Item-Skalen sind mehrheitlich gut. Die mittelstarken Binnenkorrelationen sowie die konvergenten Interkorrelationen zwischen den beiden Beurteilerperspektiven ergeben Hinweise auf die Konstruktvalidität der Stundenbogenskalen. Schlussfolgerungen: Die vorliegenden Stundenbögen bilden ein breites Spektrum psychotherapeutischer Prozesse ab und eignen sich zur Darstellung von individuellen Therapieverläufen sowie zu gruppenstatistischen Vergleichen