Additional file 1: of Do patients prefer optimistic or cautious psychiatrists? An experimental study with new and long-term patients

Data set video clips BMC Psychiatry spreadsheet. (XLS 168 kb

A Pan-<i>Lyssavirus</i> Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable <i>Rabies virus</i> and Other Lyssaviruses

<div><p>Rabies, resulting from infection by <i>Rabies virus</i> (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the <i>Lyssavirus</i> genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the <i>Lyssavirus</i> genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional <i>Lyssavirus</i> species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.</p></div

Positive clinical samples from human (ante mortem and postmortem) and animal (postmortem) specimen.

<p>Positive clinical samples from human (ante mortem and postmortem) and animal (postmortem) specimen.</p

List of primers and probes used in this study.

<p>List of primers and probes used in this study.</p

<i>Rabies</i> virus positive samples used to validate the sensitivity of the LN34 real-time RT-PCR assay.

<p><i>Rabies</i> virus positive samples used to validate the sensitivity of the LN34 real-time RT-PCR assay.</p

An inter- laboratory proficiency testing exercise for rabies diagnosis in Latin America and the Caribbean

<div><p>The direct fluorescent antibody test (DFA), is performed in all rabies reference laboratories across Latin America and the Caribbean (LAC). Despite DFA being a critical capacity in the control of rabies, there is not a standardized protocol in the region. We describe the results of the first inter-laboratory proficiency exercise of national rabies laboratories in LAC countries as part of the regional efforts towards dog-maintained rabies elimination in the American region. Twenty three laboratories affiliated to the Ministries of Health and Ministries of Agriculture participated in this exercise. In addition, the laboratories completed an online questionnaire to assess laboratory practices. Answers to the online questionnaire indicated large variability in the laboratories throughput, equipment used, protocols availability, quality control standards and biosafety requirements. Our results will inform actions to improve and harmonize laboratory rabies capacities across LAC in support for the regional efforts towards elimination of dog-maintained rabies.</p></div

Sequence alignment of the LN34 assay target region from isolates of 14 <i>Lyssavirus</i> species.

<p>Sequence alignment of the LN34 assay target region from isolates of 14 <i>Lyssavirus</i> species.</p

Scatterplot of Sensitivity by Specificity.

<p>The sensitivity (y-axis), or the true positive rates and the specificity (x-axis), or the true negative rates, for each laboratory are shown with an open circle. A laboratory correctly identifying all samples would be found in the upper right corner.</p

Selected questions of the rabies laboratory practices questionnaire and results.

<p>Selected questions of the rabies laboratory practices questionnaire and results.</p

Agreement between Laboratory and CDC values, by Sample.

<p>Agreement between Laboratory and CDC values, by Sample.</p