RESUMO
This study was performed to comparably assess two commercial real-time PCR assays for the identification of Trypanosoma cruzi DNA in serum. A total of 518 Colombian serum samples with high pre-test probability for infections with either T. cruzi or apathogenic Trypanosoma rangeli were assessed. The assessment comprised the NDO real-time PCR (TIB MOLBIOL, ref. no. 53-0755-96, referred to as the TibMolBiol assay in the following) with specificity for T. cruzi and the RealStar Chagas PCR Kit 1.0 (altona DIAGNOSTICS, order no. 611013, referred to as the RealStar assay in the following) targeting a kinetoplast sequence of both T. cruzi and T. rangeli without further discrimination. To discriminate between T. cruzi- and T. rangeli-specific real-time PCR amplicons, Sanger sequencing results were available for a minority of cases with discordant real-time PCR results, while the amplicons of the remaining discordant samples were subjected to nanopore sequencing. The study assessment indicated a proportion of 18.1% (n = 94) T. cruzi-positive samples next to 24 samples (4.6%) containing DNA of the phylogenetically related but apathogenic parasite T. rangeli. The observed diagnostic accuracy as expressed by sensitivity and specificity was 97.9% (92/94) and 99.3% (421/424) with the TibMolBiol assay and 96.8% (91/94) and 95.0% (403/424) with the RealStar assay, respectively. Reduced specificity resulted from cross-reaction with T. rangeli in all instances (3 cross-reactions with the TibMolBiol assay and 21 cross-reactions with the RealStar assay). DNA from the six discrete typing units (DTUs) of T. cruzi was successfully amplified by both real-time PCR assays. In summary, both assays showed a comparable diagnostic accuracy for the diagnosis of T. cruzi from human serum, with a slightly higher specificity seen for the TibMolBiol assay. The pronounced co-amplification of DNA from apathogenic T. rangeli according to the RealStar assay may be a disadvantage in areas of co-circulation with T. cruzi, while the test performance of the two compared assays will be quite similar in geographic settings where T. rangeli infections are unlikely.
RESUMO
Due to broad and overlapping substrate specificities, aminotransferases remain the last uncharacterized enzymes from most amino acid biosynthetic pathways in Corynebacterium glutamicum. We report here a complete description of all aminotransferases participating in the biosynthesis of the branched-chain amino acids and phenylalanine in C. glutamicum. We used methods of profile analysis on the newly available genome sequence to systematically search for and characterize members of the four known aminotransferase classes. This led to the discovery of sixteen new, potential aminotransferase encoding genes in the C. glutamicum genome, eleven of which were subsequently characterized experimentally with respect to their participation in different amino acid biosynthetic pathways. Disruption by insertion mutagenesis of ilvE, encoding a branched-chain amino acid aminotransferase, confirmed its function in leucine and isoleucine biosynthesis. Two double mutants lacking both ilvE and genes classified as class I aminotransferases exhibited additional auxotrophic requirements for valine and phenylalanine, respectively. In C. glutamicum the branched-chain amino acid aminotransferase thus participates in four amino acid biosynthetic pathways, for which in case of valine and phenylalanine biosynthesis two additional enzymes with overlapping substrate specificity exist. The novel protein with aminotransferase activity in valine biosynthesis belongs to the very recently described MocR subfamily of GntR-type helix-turn-helix transcriptional regulators, is located upstream of a potential operon of a newly described pyridoxine biosynthetic pathway and when disrupted, gives rise to a pyridoxine auxotrophy. The theoretical and experimental data we present should further provide a solid platform for ongoing research and understanding of the network of aminotransferases which participate in amino acid biosynthesis in C. glutamicum.