Load In Blood Samples Research Articles

A Bivalent Adenovirus-Vectored Vaccine Induces a Robust Humoral Response, but Does Not Protect Cynomolgus Macaques Against a Lethal Challenge With Sudan Virus.

The most recent Sudan virus (SUDV) outbreak in Uganda was first detected in September 2022 and resulted in 164 laboratory-confirmed cases and 77 deaths. There are no approved vaccines against SUDV. Here, we investigated the protective efficacy of ChAdOx1-biEBOV in cynomolgus macaques using a prime or a prime-boost regimen. ChAdOx1-biEBOV is a replication-deficient simian adenovirus vector encoding SUDV and Ebola virus (EBOV) glycoproteins (GPs). Intramuscular vaccination induced SUDV and EBOV GP-specific immunoglobulin G responses and neutralizing antibodies. Upon challenge with SUDV, vaccinated animals showed signs of disease like those observed in control animals, and no differences in survival outcomes were measured among all 3 groups. Viral load in blood samples and in tissue samples obtained after necropsy were not significantly different between groups. Overall, this study highlights the importance of evaluating vaccines in multiple animal models and demonstrates the importance of understanding protective efficacy in both animal models and human hosts.

PCR and peptide based PCMV detection in pig - development and application of a combined testing procedure differentiating newly from latent infected pigs.

Porcine cytomegalovirus (PCMV) is widely distributed in pigs and difficult to detect due to latency. PCMV infection of source pigs was associated with early graft failure after cardiac and renal xenotransplantation into nonhuman primates. Importantly, PCMV infection of the first genetically modified pig heart into a human may have contributed to the reduced survival of the patient. Sensitive and reliable assays for detection of latent PCMV infection are thus indispensable. Here, we report the development of five peptide-induced rabbit antisera specific for PCMV glycoprotein B (gB) and their validation for detection of PCMV in infected pig fallopian tube (PFT) cells by immunofluorescence and electron microscopy (EM). The anti-gB antibodies were also used for detection by Western blot analysis of PCMV purified from the supernatant of infected PFT cells. Sera of infected versus non-infected pigs have been compared. In parallel, PCMV viral load in blood samples of the animals was quantified by a novel highly sensitive nested-PCR and qPCR assay. A combination of four partly overlapping peptides from the gB C-terminus was used to establish a diagnostic ELISA for PCMV gB specific pig antibodies which is able to differentiate infected from non-infected animals and to quantify maternal antibodies in neonates. The combination of a highly sensitive nested PCR for direct virus detection with a sensitive peptide-based ELISA detecting anti-PCMV gB-antibodies, supplemented by Western blot analysis and/or immunohistochemistry for virus detection will reliably differentiate pigs with active infection, latently infected pigs, and non-infected pigs. It may significantly improve the virologic safety of xenotransplantation.

Clinical application of Epstein-Barr virus DNA loads in Epstein-Barr virus-associated diseases: A cohort study

ObjectivesThis study evaluated the diagnostic value of Epstein-Barr virus (EBV) DNA load in blood samples of patients with EBV-associated diseases, and proposed a strategy for the interpretation of positive EBV DNA results. MethodsDerivation and validation cohorts were established to evaluate the clinical significance of EBV DNA loads in the peripheral blood mononuclear cells (PBMCs) and plasma from EBV-infected patients. EBV DNA loads were compared and receiver operating characteristic curves were employed to assess the optimal cutoff values of EBV DNA for identification of EBV-associated diseases. ResultsThe derivation and validation cohorts comprised 135 and 71 subjects, respectively. EBV DNA loads in the PBMCs of the EBV-associated diseases group was significantly higher than that of the EBV non-associated diseases group (5.8 × 104 vs 7.8 × 103 copies/106 cells, P<0.0001). The diagnostic cut-off value of viral load in PBMCs for EBV-associated diseases was determined to be 1.6 × 104 copies/106 cells. The combined EBV DNA load cutoff in PBMCs and positive EBV DNA qualitative detection in plasma (>500 copies/mL) allowed for the differentiation of EBV-associated and non-associated diseases; the sensitivity and specificity were 80.6 and 96.8%, respectively. ConclusionsThe strategy of combining EBV DNA loads in PBMCs and plasma will potentially help identify EBV-associated diseases.

A sensitive and reliable quantitative immunohistochemistry technique to evaluate the percentage of Trypanosoma cruzi-infected tissue area.

Quantification of parasites in the context of Chagas disease is required to monitor the treatment with benznidazole, disease-associated cardiomyopathies and graft rejection after heart transplantation. As parasitological exams lack sensitivity, Real Time Polymerase Chain Reaction (rt-PCR) has emerged to evaluate the parasite load in blood samples and cardiac biopsies. However, despite its higher sensitivity, rt-PCR does not provide information on the location and distribution of amastigote nests within infected tissues, the characterization of inflammatory infiltrates or changes to tissue architecture. On the contrary, a sensitive immunohistochemistry technique (IHC) could fill these gaps. In the present study, a quantitative IHC exam was standardized and validated by testing adipose and cardiac tissues of experimentally infected mice containing variable parasite load levels of T. cruzi assessed by a sensitive Sybr Green rt-PCR with kDNA primers. Tissues were divided into four groups according to the parasite load: group A- 100 parasites/50ng of DNA; group B -10 parasites; group C - around 1 parasite and group D - less than 1 parasite/50ng/DNA. IHC was able to detect T. cruzi in the four groups, even in group D tissues containing fractions of a single parasite/50ng of DNA sample according to rt-PCR. In conclusion, a highly sensitivity and reliable quantitative immunohistochemistry technique was developed and is proposed to estimate the percentage of T. cruzi-infected tissue area in chagasic patients presenting with cardiomyopathies, as a complementary test to rt-PCR.

Clinical features of Epstein-Barr virus in the intestinal mucosa and blood of patients with inflammatory bowel disease.

Background:The role of Epstein–Barr virus (EBV) in inflammatory bowel disease (IBD) remains to be elucidated. The aim of this study was to investigate the presence of EBV in the blood and intestinal mucosa of patients with IBD and evaluate the association between EBV positivity and IBD.Methods:Patients with IBD, hospitalized between January 2015 and April 2018, were enrolled. The EBV-DNA load in blood samples from each subject was analyzed by quantitative real-time polymerase chain reaction. EBV-encoded small-RNA 1 (EBER-1) was detected by in-situ hybridization in intestinal mucosa tissue sections of patients with IBD.Result:EBV-DNA was detected in 48 out of 568 patients with IBD (8.4%), and EBER-1 positivity was detected in 27 of these patients (56.3%). Refractory IBD and severe mucosal inflammation were more common in patients with detectable levels of EBER-1 than in those without; the number of EBER-1-positive cells positively correlated with mucosal inflammation (P value < 0.05). Age (≥60 years old) and use of azathioprine were risk factors for EBV infection. There was no significant difference in clinical remission rate and surgical rate between the EBER-1 positive group and EBER-1 negative group, antiviral group and the non-antiviral group, among IBD patients who tested positive for EBV-DNA.Conclusion:Elderly patients with IBD, treated with azathioprine, are more susceptible to EBV positivity. Further, EBV mucosal detection correlated with the severity of mucosal damage and refractoriness, but not prognosis.

Biochips for Direct Detection and Identification of Bacteria in Blood Culture-Like Conditions

Bloodstream bacterial infections are life-threatening conditions necessitating prompt medical care. Rapid pathogen identification is essential for early setting of the best anti-infectious therapy. However, the bacterial load in blood samples from patients with bacteremia is too low and under the limit of detection of most methods for direct identification of bacteria. Therefore, a preliminary step enabling the bacterial multiplication is required. To do so, blood cultures still remain the gold standard before bacteremia diagnosis. Bacterial identification is then usually obtained within 24 to 48 hours -at least- after blood sampling. In the present work, the fast and direct identification of bacteria present in blood cultures is completed in less than 12 hours, during bacterial growth, using an antibody microarray coupled to a Surface Plasmon Resonance imager (SPRi). Less than one bacterium (Salmonella enterica serovar Enteritidis) per milliliter of blood sample is successfully detected and identified in blood volumes similar to blood tests collected in clinics (i.e. several milliliters). This proof of concept demonstrates the workability of our method for human samples, despite the highly complex intrinsic nature of unprocessed blood. Our label-free method then opens new perspectives for direct and faster bacterial identification in a larger range of clinical samples.

Biomacromolecules as tools and objects in nanometrology\u2014current challenges and perspectives

Nucleic acids, proteins, and polysaccharides are the most important classes of biopolymers. The inherent properties of biomacromolecules are contrary to those of well-defined small molecules consequently raising a number of specific challenges which become particularly apparent if biomacromolecules are treated as objects in quantitative analysis. At the same time, their specific functional ability of molecular recognition and self-organization (e.g., enzymes, antibodies, DNA) enables us to make biomacromolecules serving as molecular tools in biochemistry and molecular biology, or as precisely controllable dimensional platforms for nanometrological applications. Given the complexity of biomacromolecules, quantitative analysis is not limited to the measurement of their concentration but also involves the determination of numerous descriptors related to structure, interaction, activity, and function. Among the biomacromolecules, glycans set examples that quantitative characterization is not necessarily directed to the measurement of amount-of-substance concentration but instead involves the determination of relative proportions (molar ratios) of structural features for comparison with theoretical models. This article addresses current activities to combine optical techniques such as Raman spectroscopy with isotope dilution approaches to realize reference measurement procedures for the quantification of protein biomarkers as an alternative to mass spectrometry-based techniques. Furthermore, it is explored how established ID-MS protocols are being modified to make them applicable for quantifying virus proteins to measure the HIV viral load in blood samples. As an example from the class of carbohydrates, the challenges in accurate determination of substitution patterns are outlined and discussed. Finally, it is presented that biomacromolecules can also serve as tools in quantitative measurements of dimensions with an example of DNA origami to generate defined dimensional standards to be used for calibration in super-resolution fluorescence microscopy.Graphical abstractQuantitative analysis of biomacromolecules is accompanied with special challenges different from low molecular weight compounds. In addition, they are not only objects but also tools applicable for quantitative measurements

Diagnostic values for the viral load in peripheral blood mononuclear cells of patients with chronic active Epstein-Barr virus disease.

Chronic active Epstein-Barr virus disease (CAEBV) is a distinct EBV-associated lymphoproliferative disease with a poor prognosis. Although the viral load in blood samples has been widely used for diagnosing CAEBV, well-defined viral load thresholds to guide clinicians are currently lacking. The aim of the present study was to determine standardized diagnostic values for EBV load in blood samples of CAEBV patients using the World Health Organization international standard for reporting. Levels of EBV DNA in 103 peripheral blood mononuclear cells (PBMCs) and 95 plasma/serum samples from 107 cases with CAEBV were quantified and expressed in international units. Receiver operating characteristic curves were analyzed to assess the most appropriate cut-off values for levels of EBV DNA to distinguish CAEBV from EBV-associated infectious mononucleosis (IM) and controls with past EBV infection. Levels of EBV DNA in PBMCs were significantly higher in the CAEBV group (median, 10(4.2)IU/μgDNA) compared to the IM (median, 10(2.1)IU/μgDNA) and control groups. An inconsistent qualitative result was seen in 13 of 86 CAEBV patients; in these, EBV-DNA was positive in PBMCs, but negative in plasma. Diagnostic cut-off values for viral load in PBMCs from CAEBV patients, as compared to those of healthy controls and IM patients, were 10(2.0)IU/μgDNA and 10(3.2)IU/μgDNA, respectively. For diagnostic purposes, the viral load of PBMCs was better than of plasma/serum. A diagnostic cut-off EBV load for CAEBV may be useful for the management of CAEBV patients.

Comparison of Blood and Feather Pulp Samples for the Diagnosis of Marek's Disease and for Monitoring Marek's Disease Vaccination by Real Time-PCR

Comparison of blood and feather pulp (FP) samples for the diagnosis of Marek's disease (MD) and for monitoring Marek's diseases vaccination in chickens (serotypes 2 and 3 vaccines) by real time-PCR was evaluated. For diagnosis of MD, quantification of serotype 1 Marek's disease virus (MDV) DNA load was evaluated in 21 chickens suffering from MD. For each chicken, samples of blood and FP were collected and MDV DNA load was quantified. Solid tumors are the sample of choice for MD diagnosis by real time-PCR and, hence, 14 solid tumors were included in the study as positive controls. Load of MDV DNA in FP was equivalent to that detected in solid tumors (threshold cycle [Ct] ratio above 1.7). MDV DNA load in blood samples was lower than in solid tumors and FP samples. Nonetheless, there was a statistically significant correlation of the results obtained from FP and blood (r = 0.92). Results of the Pearson correlation test showed that Ct ratio values of 1.7 in FP correspond to Ct ratio values of 1.2 in peripheral blood. For monitoring vaccines, serotypes 2 and 3 MDV DNA load was evaluated in blood and FP samples of vaccinated chickens. Serotype 2 MDV DNA load was evaluated in samples of blood and FP from 34 chickens vaccinated with SB-1 strain. Serotype 3 MDV DNA load was evaluated in blood and FP samples from 53 chickens vaccinated with HVT strain. For both serotypes, frequency of positive samples and load of vaccine DNA was higher in FP than in blood samples. There was not a statistically significant correlation between the load of SB-1 DNA (r = 0.17) or HVT DNA (r = -0.04) in FP and blood. Our results show that the load of serotypes 1, 2, and 3 DNA is higher in FP than in blood. Diagnosis of MD could be done using both FP and blood samples. Monitoring of MD vaccination by real time-PCR required the use of FP samples. There were a high percentage of false negative samples when using blood to detect serotypes 2 and 3 MDV by real time-PCR.

A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers

BackgroundThe accurate quantification of Plasmodium falciparum parasite numbers by PCR is an important tool for monitoring growth kinetics in subjects infected and subsequently treated with anti-malarial agents.MethodsA real-time quantitative PCR (rt-qPCR) method using primers and a hydrolysis probe that targets the 18S rRNA gene was adapted and optimized to estimate parasite load in blood samples. Samples included laboratory prepared blood samples of varying parasite concentrations (6.4 × 105 to 6.4 parasites per 500 μl of packed red blood cells (500pRBC)) and blood samples collected from an experimentally infected human subject collected at 19 time points over 10 days. Sample preparation and extraction, detection chemistry, assay reproducibility, and limit of detection were compared to a previously published SYBR Green rt-qPCR used in a malaria vaccine clinical trial.ResultsBoth the rt-qPCR hydrolysis probe assay and SYBR Green rt-qPCR provided a limit of detection of 6.4 × 101 parasites per 500pRBC. However non-specific amplification in the SYBR Green rt-qPCR assay led to either inaccurate estimation of parasite load at levels below 6.4 × 102 parasites per 500pRBC and to false-positive detection of parasites in negative samples. The rt-qPCR hydrolysis probe assay was specific and provided reliable quantification of parasitaemia down to 6.4 × 101 parasites per 500pRBC. Notably, 12 of the 19 consecutive samples collected from the experimentally infected subject were at or below 6.4 × 102 copies per 500pRBC.ConclusionsThese results show that the hydrolysis probe rt-qPCR assay is superior to the SYBR Green rt-qPCR for the quantification of P. falciparum in human blood samples. The hydrolysis probe rt-qPCR is now in use in the Queensland paediatric infectious diseases laboratory (QPID) to monitor parasitaemia in experimentally-infected clinical trial subjects.

Somatic instability of the expanded GAA triplet-repeat sequence in Friedreich ataxia progresses throughout life

Friedreich ataxia (FRDA) patients are homozygous for expanded GAA triplet-repeat alleles in the FXN gene. Primary neurodegeneration involving the dorsal root ganglia (DRG) results in progressive ataxia. While it is known that DRG are inherently sensitive to frataxin deficiency, recent observations also indicate that they show age-dependent, further expansion of the GAA triplet-repeat mutation. Whether somatic instability is progressive has not been systematically investigated in FRDA patients. “Small-pool” PCR analysis of ∼2300 individual molecules from tissues of an 18-week fetus homozygous for expanded alleles revealed very low levels of instability compared with adult-derived tissues (4.2% versus 30.6%, p < 0.0001). Mutation load in blood samples from multiple patients and carriers increased significantly with age, ranging from 7.5% at 18-weeks gestation to 78.7% at 49 years of age ( R = 0.91; p = 0.0001). Therefore, somatic instability in FRDA occurs mostly after early embryonic development and progresses throughout life, lending further support to the role of postnatal somatic instability in disease pathogenesis.

Relationship of cytomegalovirus viral load in blood to pneumonitis in lung transplant recipients.

We developed a multiplex, quantitative, real-time, polymerase chain reaction assay for cytomegalovirus (CMV) and used it to measure the CMV viral load in weekly blood specimens from 43 lung transplant recipients. The median viral load in blood samples immediately preceding bronchoscopy was 1150 copies/microg human DNA for 12 subjects with pneumonitis compared to 91 copies for 31 subjects without (P=0.02, Mann-Whitney U test). Each log10 increase in CMV viral load resulted in an increase of 1.92 in the odds ratio for CMV pneumonitis (95% confidence interval 1.03-3.56). CMV viral load was elevated (>100 copies/microg human DNA) for a median of 21 days before bronchoscopy in those subjects with pneumonitis versus 0 days in those without (P=0.004). We conclude that the risk of CMV pneumonitis after lung transplantation is related to the level of CMV DNA in blood. Quantitative PCR should be evaluated prospectively for the preemptive management of CMV in lung transplant recipients.