Measurement of B-domain-deleted ReFacto AF activity with a product-specific standard is affected by choice of reagent and patient-specific factors

INTRODUCTION: Postinfusion ReFacto AF levels can be difficult to measure accurately due to discrepancies between one-stage and chromogenic FVIII assays. To overcome this, the use of the ReFacto AF laboratory standard (RAFLS) is recommended, but there are discordant reports regarding its usefulness. AIM: We investigated whether calibration with RAFLS and measurement of ReFacto AF levels are influenced by the choice of reagents and patient-specific factors in one-stage FVIII assays. METHODS: Calibration curves were generated with both the RAFLS and a plasma standard using different F8DPs and one-stage FVIII assay reagents. This selection of reagents was then used to determine FVIII levels in the plasma of patients repeatedly treated with ReFacto AF. Results were compared with those obtained with a chromogenic assay. RESULTS: F8DP devoid of von Willebrand factor (VWF) falsely increased the values of RAFLS pro-coagulant activity generated using the APTT reagent. The resulting RAFLS calibration curve underestimated ReFacto AF levels to be half of their true concentration. The use of RAFLS with F8DP containing VWF reduced the discrepancy observed between the one-stage and chromogenic FVIII assays. However, the mean difference between the two assays still varied up to 50% depending on the patient. CONCLUSIONS: The RAFLS is a suitable calibrator for one-stage FVIII assays carried out with F8DP containing VWF. However, calibration with the RAFLS does not avoid the effect of patient-specific variables that contribute to discrepancies between the measurements of ReFacto AF levels with one-stage and chromogenic FVIII assays.status: publishe

RNA-based drug susceptibility testing of Mycobacterium tuberculosis

&lt;p&gt;Background&lt;/p&gt; &lt;p&gt;Multidrug resistance of Tuberculosis strains (MDR-TB) are one of the major WHO health concerns. One of the challenges that hampers the effective response to MDR-TB is the long turnaround time of phenotypic Drug Susceptibility Testing (DST). To counter this, new fast and sensitive DNA-based methods were successfully introduced over the last years. However, these (a) are based on the knowledge on resistance mutations, (b) do not distinguish living from dead cells, (c) ignore all intrinsic resistance mechanisms, and (d) ignore the influence of compensatory mutations.&lt;/p&gt; &lt;p&gt;Objectives&lt;/p&gt; &lt;p&gt;We introduce a next-generation diagnostic test based on quantification of drug-specific RNA biomarkers. The basic principle is that a brief antibiotic exposure triggers specific transcriptional responses in susceptible, but not in resistant, microbes within a few hours. This has the advantage that long culture-dependent steps are avoided, yet the resistance phenotype is detected independent of the specific cause of resistance.&lt;/p&gt; &lt;p&gt;Materials &amp;amp; Methods&lt;/p&gt; &lt;p&gt;First, the global transcriptional response of two TB strains to 10 anti-TB drugs was determined using RNAtaq-Seq. A set of highly responsive genes was selected for each drug and RNA-targeting probes were designed.&lt;/p&gt; &lt;p&gt;Next, the RNA-based DST was developed in 96 well format. In short, 200 µl of a positively flagged MGITTM (BD) culture is spiked with a drug, while a replicate is incubated in absence of the drug. Multiplex mRNA quantification is performed directly on crude cell lysates using a combination of the bead-based MagPixTM (Luminex) and QuantigeneTM Plex (Thermo Fisher) technology. The normalized expression levels are combined to one numeric value which determines the drug susceptibility of the investigated strain.&lt;/p&gt; &lt;p&gt;Results&lt;/p&gt; &lt;p&gt;We successfully developed 8 primary sets of RNA biomarkers for ten 1st-line, 2nd-line and new drugs. Taking isoniazid as proof of principle, we present a biomarker set of 5 responsive genes and 3 normalizing genes, which enables to distinguish susceptible, low- and high resistant TB strains after 6 hours incubation. Next, preliminary results demonstrate that the biomarker sets can successfully discriminate between susceptible and resistance strains for the selected drugs.&lt;/p&gt; &lt;p&gt;Conclusion&lt;/p&gt; &lt;p&gt;We present a robust, RNA-based DST without the need for RNA extraction. The assay was proven to be efficient for isoniazid. With a total of 8 biomarker sets under optimization, the drug resistance profile of up to 14 drugs can be determined.&lt;/p&gt;</p

Non-invasive diagnosis of endometriosis based on a combined analysis of six plasma biomarkers

Lack of a non-invasive diagnostic test contributes to the long delay between onset of symptoms and diagnosis of endometriosis. The aim of this study was to evaluate the combined performance of six potential plasma biomarkers in the diagnosis of endometriosis. This case-control study was conducted in 294 infertile women, consisting of 93 women with a normal pelvis and 201 women with endometriosis. We measured plasma concentrations of interleukin (IL)-6, IL-8, tumour necrosis factor-alpha, high-sensitivity C-reactive protein (hsCRP), and cancer antigens CA-125 and CA-19-9. Analyses were done using the Kruskal-Wallis test, Mann-Whitney test, receiver operator characteristic, stepwise logistic regression and least squares support vector machines (LSSVM). Plasma levels of IL-6, IL-8 and CA-125 were increased in all women with endometriosis and in those with minimal-mild endometriosis, compared with controls. In women with moderate-severe endometriosis, plasma levels of IL-6, IL-8 and CA-125, but also of hsCRP, were significantly higher than in controls. Using stepwise logistic regression, moderate-severe endometriosis was diagnosed with a sensitivity of 100% (specificity 84%) and minimal-mild endometriosis was detected with a sensitivity of 87% (specificity 71%) during the secretory phase. Using LSSVM analysis, minimal-mild endometriosis was diagnosed with a sensitivity of 94% (specificity 61%) during the secretory phase and with a sensitivity of 92% (specificity 63%) during the menstrual phase. Advanced statistical analysis of a panel of six selected plasma biomarkers on samples obtained during the secretory phase or during menstruation allows the diagnosis of both minimal-mild and moderate-severe endometriosis with high sensitivity and clinically acceptable specificity

Performance of five rapid serological tests in mild-diseased subjects using finger prick blood for exposure assessment to SARS-CoV-2

Objectives: Assess the performance of five SARS-CoV-2 rapid serological tests (RST) using finger prick (FP) blood on-site to evaluate their usability for exposure assessment in population-based seroprevalence studies. Study design: Since cross-reactivity with common cold human coronaviruses occurs, serological testing includes a risk of false-positive results. Therefore, the selected cohort for RST-validation was based on combined immunoassay (presence of specific antibodies) and RT-qPCR (presence of SARS-CoV-2) data. RST-performance for FP blood and serum was assessed by performing each RST in two groups, namely SARSCoV-2 positive (n=108) and negative healthcare workers (n=89). Differences in accuracy and positive and negative predictive values (PPV, NPV) were calculated for a range (1-50%) of SARS-CoV-2 prevalence estimates. Results: The OrientGene showed overall acceptable performance, with sensitivities of 94.4% and 100%, and specificities of 96.6% and 94.4%, using FP blood and serum, respectively. Although three RST reach optimal specificities (100%), the OrientGene clearly outperforms in sensitivity. At a SARS-CoV-2 prevalence rate of 40%, this RST outperforms the other tests in NPV (96.3%) and reaches comparable PPV (94.9%). Although the specificity of the Covid-Presto is excellent when using FP blood or serum (100% and 97.8%, respectively), its sensitivity decreases when using FP blood (76.9%) compared to serum (98.1%). Conclusions: Performances of the evaluated RST differ largely. Only one out of five RST (OrientGene) had acceptable sensitivity and specificity using FP blood. Therefore, the latter could be used for seroprevalence studies in a high-prevalence situation. The OrientGene, which measures anti-RBD antibodies, can be valuable after vaccination as well