A novel high performing multiplex immunoassay Multi-HTLV for serological confirmation and typing of HTLV infections

BackgroundHuman T-Cell Lymphotropic Viruses (HTLV) type 1 and type 2 account for an estimated 5 to 10 million infections worldwide and are transmitted through breast feeding, sexual contacts and contaminated cellular blood components. HTLV-associated syndromes are considered as neglected diseases for which there are no vaccines or therapies available, making it particularly important to ensure the best possible diagnosis to enable proper counselling of infected persons and avoid secondary transmission. Although high quality antibody screening assays are available, currently available confirmatory tests are costly and have variable performance, with high rates of indeterminate and non-typable results reported in many regions of the world. The objective of this project was to develop and validate a new high-performance multiplex immunoassay for confirmation and discrimination of HTLV-1 and HTLV-2 strains.Methodology/Principal findingsThe multiplex platform was used first as a tool to identify suitable antigens and in a second step for assay development. With data generated on over 400 HTLV-positive blood donors sourced from USA and French blood banks, we developed and validated a high-precision interpretation algorithm. The Multi-HTLV assay demonstrated very high performance for confirmation and strain discrimination with 100% sensitivity, 98.1% specificity and 100% of typing accuracy in validation samples. The assay can be interpreted either visually or automatically with a colorimetric image reader and custom algorithm, providing highly reliable results.Conclusions/SignificanceThe newly developed Multi-HTLV is very competitive with currently used confirmatory assays and reduces considerably the number of indeterminate results. The multiparametric nature of the assay opens new avenues to study specific serological signatures of each patient, follow the evolution of infection, and explore utility for HTLV disease prognosis. Improving HTLV diagnostic testing will be critical to reduce transmission and to improve monitoring of seropositive patients.     

Identification, sequence, and expression of Treponema denticola mcpA, a putative chemoreceptor gene

The nucleotide sequence of a methyl-accepting chemotaxis protein gene, mcpA, from Treponema denticola has been determined. The mcpA gene encodes a 729-amino acid protein whose deduced amino acid sequence has significant homology with several bacterial MCPs. T. denticola McpA contains two N-terminal transmembrane regions and two C-terminal putative methylation sequences that are separated by a highly conserved signaling domain. The organization of these structural features is characteristic of MCPs. The observed molecular mass of the in vitro synthesized McpA (76.0 kDa) correlates with the predicted molecular mass of the protein (80.1 kDa).     

On the use of cross-validation for the calibration of the adaptive lasso

Cross-validation is the standard method for hyperparameter tuning, or calibration, of machine learning algorithms. The adaptive lasso is a popular class of penalized approaches based on weighted L₁-norm penalties, with weights derived from an initial estimate of the model parameter. Although it violates the paramount principle of cross-validation, according to which no information from the hold-out test set should be used when constructing the model on the training set, a “naive” cross-validation scheme is often implemented for the calibration of the adaptive lasso. The unsuitability of this naive cross-validation scheme in this context has not been well documented in the literature. In this work, we recall why the naive scheme is theoretically unsuitable and how proper cross-validation should be implemented in this particular context. Using both synthetic and real-world examples and considering several versions of the adaptive lasso, we illustrate the flaws of the naive scheme in practice. In particular, we show that it can lead to the selection of adaptive lasso estimates that perform substantially worse than those selected via a proper scheme in terms of both support recovery and prediction error. In other words, our results show that the theoretical unsuitability of the naive scheme translates into suboptimality in practice, and call for abandoning it.     

Synthesis of 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate: biological properties and potential uses.

We have synthetised 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate (in short : rATP-DNP), a derivative of ATP which carries a dinitrophenyl group. We show that rATP-DNP is a substrate for calf thymus deoxynucleotidyl terminal transferase (EC 2.7.7.31) and E. coli DNA polymerase I (Kornberg polymerase EC 2.7.7.7.). It can therefore be incorporated into DNA molecules by elongation from 3' ends or by nick translation. The incorporated dinitrophenyl group can be recognized by specific antibodies which can then be detected by anti-antibodies coupled to an enzyme. DNP groups could also be introduced into DNA after enzymatic incorporation of 8-aminohexyl adenosine 5' triphosphate and reaction with 1-fluoro-2-4-dinitrobenzene. Thus, DNA molecules carrying DNP groups can ultimately be revealed by enzymatic coloured reactions. Potential uses of this enzymatic labelling as a substitute to the radioactive detection of nucleic acids, are discussed.