Insight into Increased Recovery and Simplification of Genomic DNA Extraction Methods from Dried Blood Spots.

There is no consensus on how to perform the manual extraction of nucleic acids from dried blood spots (DBSs). Current methods typically involve agitation of the DBSs in a solution for varying amounts of time with or without heat, and then purification of the eluted nucleic acids with a purification protocol. We explored several characteristics of genomic DNA (gDNA) DBS extraction such as extraction efficiency, the role of red blood cells (RBCs) in extraction and critical kinetic factors to understand if these protocols can be simplified while maintaining sufficient gDNA recovery. We found that agitation in a RBC lysis buffer before performing a DBS gDNA extraction protocol increases yield 1.5 to 5-fold, depending upon the anticoagulant used. The use of an alkaline lysing agent along with either heat or agitation was sufficient to elute quantitative polymerase chain reaction (qPCR) amplifiable gDNA in 5 minutes. This work adds insight into the extraction of gDNA from DBSs with the intention of informing a simple, standardized manual protocol for extraction.

Quantification of psilocin in human whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

There has been burgeoning interest in psilocybin-use for the treatment of various neurological and neurodegenerative diseases. Psilocybin is mistakenly perceived as the principal pharmacologically active compound due to its high concentrations found in magic mushrooms; however, it is the prodrug of psilocin. Despite the expanding body of clinical research seeking to understand the pharmacodynamic/pharmacokinetic properties of psilocin, and its role in inducing dramatic changes to cognitive function, there has not been a corresponding increase in the development of sensitive analytical methods that can quantify psilocin in different biological fluids. Existing analytical methods have been developed using plasma, serum, and urine as the matrix of choice, but with the unknown blood-to-plasma ratio of psilocin, any pharmacokinetic conclusions drawn solely on plasma data may be misleading. Thus, the main objective of this study is to develop the first analytical method that utilizes SPE and LC-MS/MS to quantify psilocin in human whole blood. The SPE procedure yielded a high recovery efficiency (≥89%) with minimal matrix effects. The method was validated according to ANSI/ASB 036 guidelines. Linearity was between 0.7-200 ng/mL and encompassed previously reported ranges found in plasma/serum. Bias, within- and between-run precision for all quality controls met ANSI/ASB 036 acceptability criteria. Endogenous/exogenous interferences and carryover were negligible. Psilocin stability was assessed at 4°C over 48 h and was considered stable. Although a proof-of-concept study will need to be performed to characterize the method, this analytical workflow was able to detect and quantify psilocin in human whole blood at low limits of quantification.

A novel enzymatic method for isolation of plastic particles from human blood.

Microplastic particles have been detected in the human body. This study aimed to develop a blood digestion method that preserves microplastics during analysis. Acidic and alkaline reagents, commonly used for isolating plastic particles from organic materials, were tested on human blood samples and microplastics. Nitric acid, hydrochloric acid, potassium hydroxide, and sodium hydroxide were examined over time. Additionally, a pepsin-pancreatin combination was utilized for blood digestion. Light microscopy assessed digestion efficiency and particle count changes, while Raman microspectroscopy distinguished between plastic and cell debris. The acidic reagents were ineffective in removing the organic material, while alkaline reagents were effective without significant effects on microplastics. Blood digestion using pepsin and pancreatin demonstrated efficient digestion without negative consequences for the particles. While potassium hydroxide digestion is already established, novel use of the pepsin-pancreatin combination was introduced to digest human blood, indicating its potential for isolating plastic particles from tissue and human food.

Liposomes - Human phagocytes interplay in whole blood: effect of liposome design.

Nanomedicine holds immense potential for therapeutic manipulation of phagocytic immune cells. However, in vitro studies often fail to accurately translate to the complex in vivo environment. To address this gap, we employed an ex vivo human whole-blood assay to evaluate liposome interactions with immune cells. We systematically varied liposome size, PEG-surface densities and sphingomyelin and ganglioside content. We observed differential uptake patterns of the assessed liposomes by neutrophils and monocytes, emphasizing the importance of liposome design. Interestingly, our results aligned closely with published in vivo observations in mice and patients. Moreover, liposome exposure induced changes in cytokine release and cellular responses, highlighting the potential modulation of immune system. Our study highlights the utility of human whole-blood models in assessing nanoparticle-immune cell interactions and provides insights into liposome design for modulating immune responses.

Determination of tacrolimus in human whole blood in kidney transplant recipients using a rapid and specific LC-MS/MS method.

OBJECTIVE: To develop and validate an LC-M/SMS method for the determination of tacrolimus in human whole blood. METHOD: The LC-MS/MS method for the determination of tacrolimus in whole blood was developed and validated according to the guidelines. Concentrations of TAC in 100 kidney transplant patients measured by LC-MS/MS were compared with CMIA using correlation analysis and Bland-Altman plots. RESULTS: The method had a total chromatographic run time of 5 min. The calibration curves were linear over the range of 0.5-100.0 ng/mL with a lower limit of quantification of 1 ng/mL. The intra- and interday accuracy was within the range of 93.3%-109.2% and 96.0%-108.4%, respectively, with precision ranging from 0.8 to 9.4%. The mean extraction recoveries of TAC ranged from 102.6 to 107.8%. The mean concentrations of TAC in whole blood of kidney transplant patients measured by the two assays were different at 1, 3 months and all time points (p < 0.001), but no significant difference was observed at 6 months (p = 0.094). The correlation of data was good with the correlation coefficients (r2 ) of 0.7581, 0.8811, 0.8777, and 0.8077, respectively. Passing-Bablok regression analysis demonstrated good correlations with r2 values higher than 0.88 between TAC levels measured by LC-MS/MS and CMIA. Using Bland-Altman plots yielded average biases of 1.29, 0.79, 0.11, and 0.65 ng/mL at 1, 3, and 6 months and all time points. CONCLUSION: The LC-MS/MS method was validated for the accurate determination of TAC in human whole blood. The comparison of tacrolimus concentrations measured by the LC-MS/MS with CMIA showed a good correlation and agreement of two methods, suggesting LC-MS/MS should be used routinely to monitor TAC concentrations in kidney transplant patients.

Human Whole Blood Interactions with Craniomaxillofacial Reconstruction Materials: Exploring In Vitro the Role of Blood Cascades and Leukocytes in Early Healing Events.

The present study investigated early interactions between three alloplastic materials (calcium phosphate (CaP), titanium alloy (Ti), and polyetheretherketone (PEEK) with human whole blood using an established in vitro slide chamber model. After 60 min of contact with blood, coagulation (thrombin-antithrombin complexes, TAT) was initiated on all test materials (Ti > PEEK > CaP), with a significant increase only for Ti. All materials showed increased contact activation, with the KK-AT complex significantly increasing for CaP (p < 0.001), Ti (p < 0.01), and PEEK (p < 0.01) while only CaP demonstrated a notable rise in KK-C1INH production (p < 0.01). The complement system had significant activation across all materials, with CaP (p < 0.0001, p < 0.0001) generating the most pronounced levels of C3a and sC5b-9, followed by Ti (p < 0.001, p < 0.001) and lastly, PEEK (p < 0.001, p < 0.01). This activation correlated with leukocyte stimulation, particularly myeloperoxidase release. Consequently, the complement system may assume a more significant role in the early stages post implantation in response to CaP materials than previously recognized. Activation of the complement system and the inevitable activation of leukocytes might provide a more favorable environment for tissue remodeling and repair than has been traditionally acknowledged. While these findings are limited to the early blood response, complement and leukocyte activation suggest improved healing outcomes, which may impact long-term clinical outcomes.

Deep Immunophenotyping of Human Whole Blood by Standardized Multi-parametric Flow Cytometry Analyses.

Immunophenotyping is proving crucial to understanding the role of the immune system in health and disease. High-throughput flow cytometry has been used extensively to reveal changes in immune cell composition and function at the single-cell level. Here, we describe six optimized 11-color flow cytometry panels for deep immunophenotyping of human whole blood. A total of 51 surface antibodies, which are readily available and validated, were selected to identify the key immune cell populations and evaluate their functional state in a single assay. The gating strategies for effective flow cytometry data analysis are included in the protocol. To ensure data reproducibility, we provide detailed procedures in three parts, including (1) instrument characterization and detector gain optimization, (2) antibody titration and sample staining, and (3) data acquisition and quality checks. This standardized approach has been applied to a variety of donors for a better understanding of the complexity of the human immune system. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-022-00092-9.

Simultaneous determination of ten neonicotinoid insecticides and a metabolite in human whole blood by QuEChERS coupled with UPLC-Q Exactive orbitrap high-resolution mass spectrometry.

Since neonicotinoid insecticides are now the most extensively used insecticides worldwide, there are increasing cases of neonicotinoid poisoning. A rapid and sensitive method was developed for the determination of ten neonicotinoid insecticides and a metabolite 6-chloronicotinic acid in human whole blood. The types and amounts of extraction solvent, salting-out agent, and adsorbent in the QuEChERS method were optimized by comparing the absolute recoveries of 11 analytes. The separation was performed on an Agilent EC18 column with the gradient elution with 0.1% formic acid in water and acetonitrile as the mobile phase. The quantification was achieved by Q Exactive orbitrap high-resolution mass spectrometry under parallel reaction monitoring scan mode. The 11 analytes showed good linearity with R2 ≥ 0.9950, LODs ranging from 0.01 µg/L to 0.30 µg/L, and LOQs from 0.05 µg/L to 1.00 µg/L. The recoveries ranged from 78.3% to 119.9% at low, medium, and high spiked concentrations of blank blood, with matrix effects ranging from 80.9% to 117.8%, inter-day RSDs from 0.7% to 6.7%, and intra-day RSDs from 2.7% to 9.8%. The method was furthermore applied to a real case of neonicotinoid insecticide poisoning to demonstrate its feasibility. The proposed method is suitable for the rapid screening of neonicotinoid insecticides in poisoned human blood in the field of forensic science, as well as monitoring of neonicotinoid insecticide residues in humans in the field of environmental safety, compensating for a lack of studies on neonicotinoid insecticide determination in biological samples.

Disposable Electrochemical Sensors for Highly Sensitive Detection of Chlorpromazine in Human Whole Blood Based on the Silica Nanochannel Array Modified Screen-Printed Carbon Electrode.

Rapid and highly sensitive quantitative analysis of chlorpromazine (CPZ) in human whole blood is of great importance for human health. Herein, we utilize the screen-printed carbon electrodes (SPCE) as the electrode substrates for growth of highly electroactive and antifouling nanocomposite materials consisting of vertically ordered mesoporous silica films (VMSF) and electrochemically reduced graphene oxide (ErGO) nanosheets. The preparation of such VMSF/ErGO/SPCE could be performed by using an electrochemical method in a few seconds and the operation is controllable. Inner ErGO layer converted from graphene oxide (GO) in the growth process of VMSF provides oxygen-containing groups and two-dimensional π-conjugated planar structure for stable fabrication of outer VMSF layer. Owing to the π-π enrichment and excellent electrocatalytic abilities of ErGO, electrostatic preconcentration and antifouling capacities of VMSF, and inherent disposable and miniaturized properties of SPCE, the proposed VMSF/ErGO/SPCE sensor could be applied for quantitative determination of CPZ in human whole blood with high accuracy and sensitivity, good stability, and low sample consumption.

A whole blood microsampling furosemide assay: development, validation and use in a pediatric pharmacokinetic study.

Background: Furosemide is a commonly used diuretic for the treatment of edema. The pharmacokinetics of furosemide in neonates as they mature remains poorly understood. Microsampling assays facilitate research in pediatric populations. Results: We developed and validated a liquid chromatography-tandem mass spectrometry method for the quantitation of furosemide in human whole blood with volumetric absorptive microsampling (VAMS) devices (10 µl). Furosemide was stable in human whole blood VAMS under the study's assay conditions. This work established stability for furosemide for 161 days when stored as dried microsamples at -78°C. Conclusion: This method is being applied for the quantitation of furosemide in whole blood VAMS in an ongoing prospective pediatric clinical study. Representative clinical data are reported.

Bothrops jararaca Snake Venom Inflammation Induced in Human Whole Blood: Role of the Complement System.

The clinical manifestations of envenomation by Bothrops species are complex and characterized by prominent local effects that can progress to tissue loss, physical disability, or amputation. Systemic signs can also occur, such as hemorrhage, coagulopathy, shock, and acute kidney failure. The rapid development of local clinical manifestations is accompanied by the presence of mediators of the inflammatory process originating from tissues damaged by the bothropic venom. Considering the important role that the complement system plays in the inflammatory response, in this study, we analyzed the action of Bothrops jararaca snake venom on the complement system and cell surface receptors involved in innate immunity using an ex vivo human whole blood model. B. jararaca venom was able to induce activation of the complement system in the human whole blood model and promoted a significant increase in the production of anaphylatoxins C3a/C3a-desArg, C4a/C4a-desArg, C5a/C5a-desArg and sTCC. In leukocytes, the venom of B. jararaca reduced the expression of CD11b, CD14 and C5aR1. Inhibition of the C3 component by Cp40, an inhibitor of C3, resulted in a reduction of C3a/C3a-desArg, C5a/C5a-desArg and sTCC to basal levels in samples stimulated with the venom. Exposure to B. jararaca venom induced the production of inflammatory cytokines and chemokines such as TNF-α, IL-8/CXCL8, MCP-1/CCL2 and MIG/CXCL9 in the human whole blood model. Treatment with Cp40 promoted a significant reduction in the production of TNF-α, IL-8/CXCL8 and MCP-1/CCL2. C5aR1 inhibition with PMX205 also promoted a reduction of TNF-α and IL-8/CXCL8 to basal levels in the samples stimulated with venom. In conclusion, the data presented here suggest that the activation of the complement system promoted by the venom of the snake B. jararaca in the human whole blood model significantly contributes to the inflammatory process. The control of several inflammatory parameters using Cp40, an inhibitor of the C3 component, and PMX205, a C5aR1 antagonist, indicates that complement inhibition may represent a potential therapeutic tool in B. jararaca envenoming.

A Possible Way to Relate the Effects of SARS-CoV-2-Induced Changes in Transferrin to Severe COVID-19-Associated Diseases.

SARS-CoV-2 infections are responsible for the COVID-19 pandemic. Transferrin has been found to explain the link between diseases associated with impaired iron transport and COVID-19 infection. The effect of SARS-CoV-2 on human whole blood was studied by differential scanning calorimetry. The analysis of the thermal transition curves showed that the melting temperature of the transferrin-related peak decreased in the presence of SARS-CoV-2. The ratio of the under-curve area of the two main peaks was greatly affected, while the total enthalpy of the heat denaturation remained nearly unchanged in the presence of the virus. These results indicate that SARS-CoV-2, through binding to transferrin, may influence its Fe3+ uptake by inducing thermodynamic changes. Therefore, transferrin may remain in an iron-free apo-conformational state, which depends on the SARS-CoV-2 concentration. SARS-CoV-2 can induce disturbance in erythropoiesis due to toxicity generated by free iron overload.

The response of soft tissue cells to Ti implants is modulated by blood-implant interactions.

Titanium-based dental implants have been highly optimized to enhance osseointegration, but little attention has been given to the soft tissue-implant interface, despite being a major contributor to long term implant stability. This is strongly linked to a lack of model systems that enable the reliable evaluation of soft tissue-implant interactions. Current in vitro platforms to assess these interactions are very simplistic, thus suffering from limited biological relevance and sensitivity to varying implant surface properties. The aim of this study was to investigate how blood-implant interactions affect downstream responses of different soft tissue cells to implants in vitro, thus taking into account not only the early events of blood coagulation upon implantation, but also the multicellular nature of soft tissue. For this, three surfaces (smooth and hydrophobic; rough and hydrophobic; rough and hydrophilic with nanostructures), which reflect a wide range of implant surface properties, were used to study blood-material interactions as well as cell-material interactions in the presence and absence of blood. Rough surfaces stimulated denser fibrin network formation compared to smooth surfaces and hydrophilicity accelerated the rate of blood coagulation compared to hydrophobic surfaces. In the absence of blood, smooth surfaces supported enhanced attachment of human gingival fibroblasts and keratinocytes, but limited changes in gene expression and cytokine production were observed between surfaces. In the presence of blood, rough surfaces supported enhanced fibroblast attachment and stimulated a stronger anti-inflammatory response from macrophage-like cells than smooth surfaces, but only smooth surfaces were capable of supporting long-term keratinocyte attachment and formation of a layer of epithelial cells. These findings indicate that surface properties not only govern blood-implant interactions, but that this can in turn also significantly modulate subsequent soft tissue cell-implant interactions.

Discovery and quantification of plastic particle pollution in human blood.

Plastic particles are ubiquitous pollutants in the living environment and food chain but no study to date has reported on the internal exposure of plastic particles in human blood. This study's goal was to develop a robust and sensitive sampling and analytical method with double shot pyrolysis - gas chromatography/mass spectrometry and apply it to measure plastic particles ≥700 nm in human whole blood from 22 healthy volunteers. Four high production volume polymers applied in plastic were identified and quantified for the first time in blood. Polyethylene terephthalate, polyethylene and polymers of styrene (a sum parameter of polystyrene, expanded polystyrene, acetonitrile butadiene styrene etc.) were the most widely encountered, followed by poly(methyl methacrylate). Polypropylene was analysed but values were under the limits of quantification. In this study of a small set of donors, the mean of the sum quantifiable concentration of plastic particles in blood was 1.6 µg/ml, showing a first measurement of the mass concentration of the polymeric component of plastic in human blood. This pioneering human biomonitoring study demonstrated that plastic particles are bioavailable for uptake into the human bloodstream. An understanding of the exposure of these substances in humans and the associated hazard of such exposure is needed to determine whether or not plastic particle exposure is a public health risk.

Platelet-Depletion of Whole Blood Reveals That Platelets Potentiate the Release of IL-8 From Leukocytes Into Plasma in a Thrombin-Dependent Manner.

Objective: In a recent study, we found an elevated level of interleukin 8 (IL-8) in response to bacterial incubation in thrombin-sufficient human whole blood anticoagulated by the fibrin polymerization blocking peptide GPRP. Whether thrombin directly activated leukocytes or mediated the release via thrombin-dependent activation of platelets remains unresolved. Herein, we addressed the role of thrombin and platelets in IL-8 release. Methods: We separated platelets from whole blood using a combination of 0.7% (w/v) citrate and GPRP for attenuating the hemostatic response during the separation of platelets. Cytokine responses were compared in whole blood and platelet-depleted blood upon Escherichia coli incubation. Cytokine responses were also profiled with and without reconstitution of either platelets or the supernatant from activated platelets. Results: Platelets were not activated during the separation process but responded to stimuli upon re-calcification. Plasma levels of IL-1ß, IL-1Ra, IL-6, IL-8, IP-10, MIP-1α, and MIP-1ß were significantly reduced in platelet-depleted blood compared to whole blood, but recovered in the presence of platelets, or with the supernatant of activated platelets. The leukocyte fraction and platelets were each found to contribute to the elevation of IL-8 at around 5 ng/ml; however, if combined, the release of IL-8 increased to 26 ng/ml. This process was dependent on thrombin since the levels of IL-8 remained at 5 ng/ml in whole blood if thrombin was blocked. Intracellular staining revealed that monocytes were the main source for IL-8 expression. Conclusion: Our findings suggest that the release of IL-8 is mediated by the leukocytes, mainly monocytes, but potentiated via thrombin-dependent activation of platelets.

Plasma Corona Protects Human Immune Cells from Structurally Nanoengineered Antimicrobial Peptide Polymers.

Safe and effective antimicrobials are needed to combat emerging antibiotic-resistant bacteria. Structurally nanoengineered antimicrobial peptide polymers (termed SNAPPs) interact with bacterial cell membranes to potently kill bacteria but may also interact at some level with human cell membranes. We studied the association of four different SNAPPs with six different white blood cells within fresh whole human blood by flow cytometry. In whole human blood, SNAPPs had detectable association with phagocytic cells and B cells, but not natural killer and T cells. However, without plasma proteins and therefore no protein corona on the SNAPPs, a greater marked association of SNAPPs with all white blood cell types was detected, resulting in cytotoxicity against most blood cell components. Thus, the formation of a protein corona around the SNAPPs reduced the association and prevented human blood cell cytotoxicity of the SNAPPs. Understanding the bio-nano interactions of these SNAPPs will be crucial to ensuring that the design of next-generation SNAPPs and other promising antimicrobial nanomaterials continues to display high efficacy toward antibiotic-resistant bacteria while maintaining a low toxicity to primary human cells.

Influence of Poly(ethylene glycol) Molecular Architecture on Particle Assembly and Ex Vivo Particle-Immune Cell Interactions in Human Blood.

Poly(ethylene glycol) (PEG) is widely used in particle assembly to impart biocompatibility and stealth-like properties in vivo for diverse biomedical applications. Previous studies have examined the effect of PEG molecular weight and PEG coating density on the biological fate of various particles; however, there are few studies that detail the fundamental role of PEG molecular architecture in particle engineering and bio-nano interactions. Herein, we engineered PEG particles using a mesoporous silica (MS) templating method and investigated how the PEG building block architecture impacted the physicochemical properties (e.g., surface chemistry and mechanical characteristics) of the PEG particles and subsequently modulated particle-immune cell interactions in human blood. Varying the PEG architecture from 3-arm to 4-arm, 6-arm, and 8-arm generated PEG particles with a denser, stiffer structure, with increasing elastic modulus from 1.5 to 14.9 kPa, inducing an increasing level of immune cell association (from 15% for 3-arm to 45% for 8-arm) with monocytes. In contrast, the precursor PEG particles with the template intact (MS@PEG) were stiffer and generally displayed higher levels of immune cell association but showed the opposite trend-immune cell association decreased with increasing PEG arm numbers. Proteomics analysis demonstrated that the biomolecular corona that formed on the PEG particles minimally influenced particle-immune cell interactions, whereas the MS@PEG particle-cell interactions correlated with the composition of the corona that was abundant in histidine-rich glycoproteins. Our work highlights the role of PEG architecture in the design of stealth PEG-based particles, thus providing a link between the synthetic nature of particles and their biological behavior in blood.

A streamlined whole blood CyTOF workflow defines a circulating immune cell signature of COVID-19.

Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining, and data acquisition protocols can all introduce technical variation that can confound integrative analyses of large data-sets of samples processed across multiple labs. Here, we present a streamlined whole blood CyTOF workflow which addresses many of these sources of experimental variation and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We apply this workflow to characterize 184 whole blood samples collected longitudinally from a cohort of 72 hospitalized COVID-19 patients and healthy controls, highlighting dynamic disease-associated changes in circulating immune cell frequency and phenotype.

Characterization of Yersinia pestis Phage Lytic Activity in Human Whole Blood for the Selection of Efficient Therapeutic Phages.

The global increase in multidrug-resistant (MDR) pathogenic bacteria has led to growing interest in bacteriophage ("phage") therapy. Therapeutic phages are usually selected based on their ability to infect and lyse target bacteria, using in vitro assays. In these assays, phage infection is determined using target bacteria grown in standard commercial rich media, while evaluation of the actual therapeutic activity requires the presence of human blood. In the present work, we characterized the ability of two different Yersinia pestis lytic phages (ϕA1122 and PST) to infect and kill a luminescent Y. pestis EV76 strain suspended in Brain Heart Infusion (BHI)-rich medium or in human whole blood, simulating the host environment. We found that the ability of the phages to infect and lyse blood-suspended Y. pestis was not correlated with their ability to infect and lyse BHI-suspended bacteria. While the two different phages exhibited efficient infective capacity in a BHI-suspended culture, only the PST phage showed efficient lysis ability against blood-suspended bacteria. Therefore, we recommend that for personalized phage therapy, selection of phage(s) for efficient treatment of patients suffering from MDR bacterial infections should include prior testing of the candidate phage(s) for their lysis ability in the presence of human blood.

Longitudinal Variations of M. tuberculosis-Induced IFN-γ Responses in HIV-Negative Pregnant Women Exposed to Tuberculosis.

Introduction: Pregnancy triggers an alteration of the immune functions and increases the risk of developing the active tuberculosis (TB) symptoms in exposed women. The effect of pregnancy on the Mycobacterium tuberculosis-specific immune responses used for most of the TB immunodiagnostic assays is not well documented. Here we investigated the changes in the M. tuberculosis-specific IFN-γ production in age-matched pregnant and non-pregnant women according to their TB exposition status. Methods: We conducted a prospective cohort study on HIV-seronegative pregnant and non-pregnant women with compatible pulmonary TB symptoms addressed to TB healthcare facilities in Antananarivo, Madagascar. Active pulmonary TB was bacteriologically assessed with culture from sputum samples. Clinical data and blood samples were collected at inclusion and after 6 months of follow-up for each individual included. Whole blood samples were stimulated with QuantiFERON TB-Gold Plus (QFT-P) assay antigens. Plasma IFN-γ concentrations were then assessed by ELISA. Results: A total of 284 women were investigated for the study including 209 pregnant women without confirmed TB (pNTB), 24 pregnant women with bacteriologically confirmed active TB (pATB), 16 non-pregnant women with active TB (ATB), and 35 non-pregnant healthy donors (HC). At inclusion, IFN-γ responses are lower in the pregnant women compared to their age-matched non-pregnant counterparts and independently of their TB status. Among the pregnant women, higher concentrations of M. tuberculosis-specific IFN-γ were observed in those exposed to TB, but with a lower magnitude in the active TB compared to the latently infected pregnant women (p < 0.05 with TB1 and p < 0.01 with TB2). After 6 months of follow-up, the M. tuberculosis-specific IFN-γ responses return to their baseline concentrations except for the pregnant women treated for TB for which none of the QFT-P positive reversed to negative (0%, 0/10) at the end of their TB treatment. Conclusion: These results support the concept of specific immune priorities characterized by a concomitant reduction in inflammatory immunity during pregnancy and corroborate the important role of activating the M. tuberculosis-specific immune responses to control the infection when the pregnant women are exposed to the pathogen.