Molecular detection of SARS-COV-2 in exhaled breath at the point-of-need.

The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It assesses contagiousness directly, the sample is easy and comfortable to obtain, sampling can be standardized, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an RT-qPCR molecular assay to detect SARS-CoV-2 shedding. Our portable, silicon impactor allowed for the efficient capture (>85%) of respiratory particles down to 300 nm without the need for additional equipment. We demonstrate using both conventional off-chip and in-situ PCR directly on the silicon chip that sampling subjects' breath in less than a minute yields sufficient viral RNA to detect infections as early as standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test remains positive during the first week but is the first to consistently report a negative result, putatively signalling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections.

Exhaled breath SARS-CoV-2 shedding patterns across variants of concern.

OBJECTIVES: We performed exhaled breath (EB) and nasopharyngeal (NP) quantitative polymerase chain reaction (qPCR) and NP rapid antigen testing (NP RAT) of SARS-CoV-2 infections with different variants. METHODS: We included immuno-naïve alpha-infected (n = 11) and partly boosted omicron-infected patients (n = 8) as high-risk contacts. We compared peak NP and EB qPCR cycle time (ct) values between cohorts (Wilcoxon-Mann-Whitney test). Test positivity was compared for three infection phases using Cochran Q test. RESULTS: Peak median NP ct was 11.5 (interquartile range [IQR] 10.1-12.1) for alpha and 12.2 (IQR 11.1-15.3) for omicron infections. Peak median EB ct was 25.2 (IQR 24.5-26.9) and 28.3 (IQR 26.4-30.8) for alpha and omicron infections, respectively. Distributions did not differ between cohorts for NP (P = 0.19) or EB (P = 0.09). SARS-CoV-2 shedding peaked on day 1 in EB (confidence interval [CI] 0.0 - 4.5) and day 3 in NP (CI 1.5 - 6.0). EB qPCR positivity equaled NP qPCR positivity on D0-D1 (P = 0.44) and D2-D6 (P = 1.0). It superseded NP RAT positivity on D0-D1 (P = 0.003) and D2-D6 (P = 0.008). It was inferior to both on D7-D10 (P < 0.001). CONCLUSION: Peak EB and nasopharynx shedding were comparable across variants. EB qPCR positivity matched NP qPCR and superseded NP RAT in the first week of infection.

Acquired functional coagulation inhibitors: review on epidemiology, results of a wet-workshop on laboratory detection, and implications for quality of inhibitor diagnosis.

The accurate detection and quantification of coagulation inhibitors remains a challenging problem for most diagnostic laboratories. Prolonged screening assays and abnormal results of mixing tests with normal plasma may indicate the presence of such inhibitors. Yet, the presence of lupus anticoagulant, heparin, and potential contamination of plasma with therapeutically active antithrombotic drugs has to also be ruled out. This review covers the epidemiology of acquired functional coagulation inhibitors, and reports the results of a wet-workshop, organized by the External Quality Control for Assays and Test (ECAT) Foundation, on laboratory detection of such inhibitors. The aim of the workshop was to investigate, within groups of experts from dispersed professional laboratories, the quality of inhibitor detection and the difficulties encountered during the analytical process. In this workshop 8 samples representing varying milieu were tested by 10 groups of participants from 20 different countries. Workshop participants were asked to report the results of all investigations performed and to provide a likely diagnosis and/or a conclusion of the hemostasis abnormality represented by the test samples. Generally, the sensitivity of inhibitor detection was high but the differential diagnosis of the type of inhibitors identified was unsatisfactory, as many false-positive and false-negative results were observed. The most remarkable observation was the lack of a clear step-by-step analysis of the nature of an inhibitor once a positive mixing test had been detected. The possible consequences of these observations for the appropriate diagnosis and clinical management of patients are outlined. A diagnostic algorithm for the differential diagnosis and confirmation of acquired coagulation inhibitors is presented.

Real-time monitoring of DNA hybridization and melting processes using a fiber optic sensor.

In this paper a fiber optic surface plasmon resonance (FO-SPR) sensor was used to analyze the melting process of DNA linked to silica nanoparticles. Real-time monitoring of a DNA melting process has rarely been studied using surface plasmon resonance (SPR), since most commercial SPR setups do not allow for dynamic and accurate temperature control above 50 °C. The FO-SPR sensor platform, with silica nanobead signal amplification, allows sensing inside a standard PCR thermocycler, which makes high resolution DNA melting curve analysis possible. This innovative combination was used to characterize the hybridization and melting events between DNA immobilized on the sensor surface and DNA probes on silica nanoparticles. At optimized hybridization conditions complementary DNA strands of different lengths could be distinguished. While the real-time FO-SPR analysis of DNA hybridization did not result in significant variances, the analysis of DNA melting determined the exact length of overlap and the matching Gibbs energy.

A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis.

Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and emission spectra. The assay was tested in vitro using modulators like heparin, hirudin, epsilon-aminocaproic acid, gly-pro-arg-pro peptide and reptilase and validated by measurement of prothrombin fragment 1+2 and plasmin-alpha2-antiplasmin levels. Intra- and inter-assay coefficients of variation were < 9% and 6-25%, respectively. Interplay between coagulation and fibrinolysis was demonstrated by the effect of tissue-type plasminogen activator on thrombin generation and by the different responses of activated protein C and thrombomodulin on fibrinolysis. The last responses showed the linkage between coagulation and fibrinolysis by thrombin activatable fibrinolysis inhibitor. In conclusion, this strategy allows detection of coagulation, fibrinolysis and their interplay in a single assay.

Thrombocytopenia in early malaria is associated with GP1b shedding in absence of systemic platelet activation and consumptive coagulopathy.

Thrombocytopenia develops early in malaria, but the underlying mechanisms remain incompletely understood. We studied the aetiology of malaria-associated thrombocytopenia in volunteers experimentally infected with Plasmodium falciparum malaria, in Indonesian malaria patients and in ex vivo studies. In experimental human malaria, the decrease in platelet counts was associated with a concurrent rise in young platelets (immature platelet fraction) and thrombopoietin. D-dimer concentrations were moderately elevated without a prolongation in the activated partial thromboplastin time or decrease in fibrinogen. There was no increase in expression of the platelet surface markers CD62P, PAC-1 and CD63 and in plasma concentrations of the platelet factors P-selectin, CXCR4, CXCL7, RANTES and CD40L. In contrast, concentrations of soluble glycoprotein-1b (sGP1b), the external domain of the platelet receptor for von Willebrand factor (VWF), increased early. Indonesian malaria patients also had elevated concentrations of sGP1b, which correlated with VWF concentrations. Finally, incubation of platelets with parasitized erythrocytes in vitro failed to induce platelet aggregation or activation. We concluded that neither compromised platelet production nor platelet activation or consumptive coagulopathy were responsible for the early thrombocytopenia in malaria. We hypothesize that the increase in sGP1b concentrations results from VWF-mediated GP1b shedding; a process that may prevent excessive adhesion of platelets and parasitized erythrocytes.

Improved method for counting DNA molecules on biofunctionalized nanoparticles.

In order to accurately determine low numbers (1-100) of immobilized ssDNA molecules at a single, silica 250 nm nanoparticle surface, we hereby propose an integrated approach combining classic single molecule confocal microscopy (SMCM), that is, stepwise photobleaching of labeled ssDNA, with modified total internal reflection fluorescence microscopy (mTIRF). We postulate that SMCM alone is unable to exactly account for all labeled ssDNA because of inherent laser polarization effects; that is, perpendicularly oriented molecules to the sample surface are not (or are only slightly) susceptible to laser excitation and thus are invisible in a classic photobleaching experiment. The SMCM method accounts for at best two-thirds (68%) of the present ssDNA molecules. The principle of the mTIRF technique, which relies on the creation of highly inclined illumination combined with part of the laser remaining in normal Kohler illumination, enables accurate counting of SMCM invisible molecules. The combined approach proposed here circumvents the polarization issue and allows a complete single molecule counting on individual nanoparticles, fully in line with bulk measurements, as will be demonstrated.

The between-laboratory variation of factor VIII inhibitor testing: the experience of the external quality assessment program of the ECAT foundation.

The detection and quantification of factor VIII (FVIII) inhibitors is clinically important both for the identification of hemophilia A patients with inhibitors and for the management of immune tolerance treatment. Only limited data are available on the between-laboratory variation of FVIII inhibitor testing. This report describes the evaluation of the results of the large-scale external quality assessment program of the European Concerted Action on Thrombosis Foundation. This study includes the results of six different surveys for the period 2006 to 2008 with 100 to 170 participating laboratories. The overall between-laboratory variation ranged from 28% to 52% with a slightly lower variation for the Nijmegen assay (approximately 39%) on average than for the Bethesda assay (approximately 45%). The use of buffered normal pooled plasma as FVIII source showed better performance compared with the use of nonbuffered pooled plasma; likewise the use of FVIII-deficient plasma compared with the use of imidazole buffer. However, the combination of both was essential for lowest between-laboratory variation. The Nijmegen assay also showed better performance with respect to specificity and sensitivity than the Bethesda assay, although the results for neither were entirely satisfactory. In general, it can be concluded that the measurement of FVIII inhibitory antibodies with the Nijmegen assay should be favored over the use of the Bethesda assay. However, further improvement of the laboratory test for FVIII inhibitors is urgently needed.

Improvements in factor VIII inhibitor detection: From Bethesda to Nijmegen.

All methods for the detection of factor VIII (FVIII) inhibitors are based on the measurement of inactivation of FVIII in a mixture of the test plasma containing the putative inhibitor and an exogenous source of FVIII. Various types of assays have been developed since the first inhibitor was described in 1941. Nowadays, two methods are preferably used, the Bethesda assay and the Nijmegen assay. Although the Nijmegen assay shows a better specificity and intra- and interlaboratory variation, it is still hampered by several limitations related to assay characteristics (pH, temperature, and time of incubation), type of control sample, and the von Willebrand factor content of the assay mixture. Epitope specificity plays an important role in the reliability of functional assays because inhibitors against the C2 domain are more difficult to quantify compared with inhibitors against the A2 domain. Finally, lupus anticoagulants can interfere with inhibitor assays, resulting in aberrant results. This report describes in detail the various problems encountered with the assays used in the quantification of functional FVIII inhibitors.

Use of the platelet function analyzer to minimize bleeding complications after renal biopsy.

BACKGROUND: The bleeding time is frequently used to screen primary haemostasis before surgical procedures, although it poorly predicts the risk of hemorrhage. The platelet function analyzer (PFA), which is also used to screen primary haemostasis, has a higher sensitivity and other advantages, like patient friendliness, higher degree of objectivity and analytical reliability, but needs more extensive clinical validation. METHODS: We compared the predictive values of the PFA-CTs (closure times) and bleeding time for bleeding events after renal biopsy. We prospectively evaluated the complications in patients that underwent a renal biopsy and were screened with PFA in advance (n=170). For comparison we used a historical cohort of patients screened with the bleeding time (n=132). RESULTS: When the PFA-CTs were normal, 26.0% of the patients had a mild bleeding event after the biopsy, which did not differ from the event rate with a normal bleeding time (29.4%). When one or both PFA-CTs were prolonged, 51.3% of the patients had post-biopsy bleeding events independently of the measures to correct the closure time(s), significantly more than with either a prolonged bleeding time (26.7%) or normal PFA-CTs (26.0%). CONCLUSION: For bleeding events, the PFA has a higher positive and similar negative predictive value compared to the bleeding time. Taken into account the additional advantages of the PFA like patient friendliness and better analytical qualities, we prefer the PFA over the bleeding time as a screening tool for primary haemostasis before performing a renal biopsy.

A model for the harmonisation of test results of different quantitative D-dimer methods.

The numerical test results of different D-dimer assays vary widely. Because of the complexity of the analyte of target as well as the variability in specificity of different D-dimer assays, only harmonisation of the test results seems to be feasible. The use of a single conversion factor does not take into account for several methods the lack of commutability between test results and consensus values at different D-dimer levels. This is probably related to the mutually different response of methods to high and low levels. We therefore designed a harmonisation model based on the transformation of a method-specific regression line to a reference regression line. We used the data for the measurement of a set of plasma samples with different D-dimer levels by 353 different laboratories using 7 of the most frequently used quantitative D-dimer methods. For each method we calculated the method-specific consensus value for each sample. The overall median value was also estimated. Per method linear regression was applied throughout the method-specific consensus values using the amount of patient pooled plasma added to the different plasma samples as the independent variable. The line through the overall median values of all 7 methods was used as the reference line. Harmonisation between the methods was obtained by transformation of the method-specific regression line to the reference line. This harmonisation resulted in a reduction of the variability between the method-specific consensus values from about 75% to about 5.5%. Clinical validation of this concept had shown significant improvement of the test result comparability. We conclude that this model is a feasible approach in the harmonisation of D-dimer methods. If the harmonisation procedure is included in the calibration procedure by the manufacturers, customers will automatically obtain harmonised test results.

Thirteen novel mutations in the factor VIII gene in the Nijmegen haemophilia A patient population.

The development of neutralising antibodies to factor VIII (FVIII) is a major complication of haemophilia A (HA) therapy. We aimed to construct an individual risk profile for the development of inhibitors in HA and started by screening for the causative mutation in our HA patient population. A total of 109 patients and 28 carriers were screened. The analysis revealed 38 different mutations in the FVIII gene, of which 13 have not been described on the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS). Twenty-five mutations have been reported previously and all except two had a similar phenotype to what has been described. Three novel mutations were associated with severe HA: one non-missense mutation, a small insertion in the A2 domain, and two missense mutations, a H256R mutation in the A1 domain and a L2025P substitution in the C1 domain. One novel mutation, Y156C, was associated with moderate HA. Nine novel mutations caused mild HA. The P130R, D167E and V278M mutations are located in the A1 domain. R439C, Y511H, A544G and Q645H in the A2 domain, L1758F in the A3 domain and a S2157R mutation in the C1 domain. In conclusion, the genotypic profile of our HA population was not different from others described and is suitable to study inhibitor formation.

Effect of homocysteine reduction by B-vitamin supplementation on markers of clotting activation.

Homocysteine may have an effect on risk of cardiovascular disease by stimulating procoagulant factors and/or impair anti-coagulant mechanisms or fibrinolysis. However, data in humans of such effects are sparse. In this intervention study, we examined the effect of homocysteine lowering by B-vitamin supplementation on prothrombin fragments 1 and 2 (F1 + 2), thrombin-antithrombin complex (TAT), and fibrin degradation products (D-dimer). The study comprised 118 healthy volunteers, 50 with homocysteine > 16 mumol/L and 68 with homocysteine < or = 16 mumol/L, who were randomized to placebo or high-dose B-vitamin supplements (5 mg folic acid, 0.4 mg hydroxycobalamin, and 50 mg pyridoxine) daily for 8 weeks. Although homocysteine concentrations were 27.7% (p < 0.0001) reduced in the B-vitamin group compared to the placebo group, no effect on F1 + 2 and TAT concentrations was observed. A 10.4% reduction was observed for D-dimer (p = 0.08). In conclusion, it appears that in healthy subjects homocysteine reduction by B-vitamin supplementation has a modest beneficial effect on clotting activation.