Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

African swine fever virus (ASFV) is the causative agent of a deadly disease in pigs and is spread rapidly across borders. Samples collected from suspected cases must be sent to the reference laboratory for diagnosis using polymerase chain reaction (PCR). In this study, we aimed to develop a simple DNA isolation step and real-time recombinase polymerase amplification (RPA) assay for rapid detection of ASFV. RPA assay based on the p72 encoding B646L gene of ASFV was established. The assays limit of detection and cross-reactivity were investigated. Diagnostic performance was examined using 73 blood and serum samples. Two extraction approaches were tested: silica-column-based extraction method and simple non-purification DNA isolation (lysis buffer and heating, 70 °C for 20 min). All results were compared with well-established real-time PCR. In a field deployment during a disease outbreak event in Uganda, 20 whole blood samples were tested. The assay’s analytical sensitivity was 3.5 DNA copies of molecular standard per µL as determined by probit analysis on eight independent assay runs. The ASFV RPA assay only detected ASFV genotypes. Compared to real-time PCR, RPA diagnostic sensitivity and specificity were 100%. Using the heating/lysis buffer extraction procedure, ASFV-RPA revealed better tolerance to inhibitors than real-time PCR (97% and 38% positivity rate, respectively). In Uganda, infected animals were identified before the appearance of fever. The ASFV-RPA assay is shown to be as sensitive and specific as real-time PCR. Moreover, the combination of the simple extraction protocol allows its use at the point of need to improve control measures.     

Malaria PCR Detection in Cambodian Low-Transmission Settings: Dried Blood Spots versus Venous Blood Samples

In the context of malaria elimination, novel strategies for detecting very low malaria parasite densities in asymptomatic individuals are needed. One of the major limitations of the malaria parasite detection methods is the volume of blood samples being analyzed. The objective of the study was to compare the diagnostic accuracy of a malaria polymerase chain reaction assay, from dried blood spots (DBS, 5 μL) and different volumes of venous blood (50 μL, 200 μL, and 1 mL). The limit of detection of the polymerase chain reaction assay, using calibrated Plasmodium falciparum blood dilutions, showed that venous blood samples (50 μL, 200 μL, 1 mL) combined with Qiagen extraction methods gave a similar threshold of 100 parasites/mL, ∼100-fold lower than 5 μL DBS/Instagene method. On a set of 521 field samples, collected in two different transmission areas in northern Cambodia, no significant difference in the proportion of parasite carriers, regardless of the methods used was found. The 5 μL DBS method missed 27% of the samples detected by the 1 mL venous blood method, but most of the missed parasites carriers were infected by Plasmodium vivax (84%). The remaining missed P. falciparum parasite carriers (N = 3) were only detected in high-transmission areas.     

Experimental Investigation on Mechanical Properties of Geocell Strips at Low Temperature

Geocell is widely used in the treatment of poor roadbed, which can restrain soil laterally and improve the stability of soil. In cold area engineering, a change in temperature can influence the mechanical properties of geocell of different materials. To study the mechanical response of geocell at low temperatures, three types of geocell strips commonly used in engineering, namely the polyethylene (HDPE), polypropylene (PP), and polyester (PET), were studied via the uniaxial tensile test at the ambient temperatures of −5 °C, −20 °C, and −35 °C, respectively. Meanwhile, the tensile strength, fracture mode, and temperature sensitivity of geocell specimens were compared. It is concluded that: (1) at low temperatures, the tensile strengths of HDPE and PET geocell strips are significantly improved, while that of the PP geocell strip is less sensitive to the temperature. (2) The PP geocell is subject to a brittle failure at all ambient temperatures. The PET geocell strip experiences a hard-ductile failure at normal temperatures of −5 °C and −20 °C. While in the tensile test at −35 °C, it is prone to brittle failure and hard-ductile failure. The HDPE geocell strip suffers from ductile failure at all ambient temperatures. (3) At low temperatures, overall, the tensile properties of the PET geocell strip is better than those of the PP and HDPE geocell strips.     

Estimation of the Stability of Parathyroid Hormone when Stored at ?80°C for a Long Period

Background and objectives: Stability of parathyroid hormone (PTH) at −80°C for long storage periods has never been studied. This can be of importance for the conclusions of studies where blood banks have been constituted. The study''s aim was to evaluate stability of PTH when stored as serum or plasma EDTA samples at −80°C.Design, setting, participants & measurements: Samples were collected from 16 chronic hemodialysis patients using EDTA and gel-separator tubes. Plasma and serum were aliquoted; one aliquot was assayed with Elecsys and Liaison methods to determine the “baseline” values and another aliquot after 1, 3, 6, and 12 mo. The factors “method,” “tubes,” “subjects,” and “time” were included in a mixed linear model to evaluate their effects on measured PTH values. The prediction interval methodology was used to assess where a future result could be obtained with a defined probability.Results: With the Liaison method, the maximum storage times with either dry or EDTA tubes were estimated to be 9 and 2 mo, respectively. With the Elecsys method, samples could be stored at least 2 yr with acceptable level of degradation.Conclusion: PTH stability at −80°C is not infinite. Maximum storage time and acceptance limits (30%) were defined, showing that with one method, samples should be stored for not more than 2 mo, whereas the other could be stored for up to 2 yr. With any PTH assay, the maximum storage time should be evaluated to ascertain that samples will keep their initial reactive profile after prolonged storage periods.Parathyroid hormone (parathormone [PTH]) determination is routinely performed in laboratories for the diagnosis and management of renal osteodystrophy (ROD). Worldwide, nephrologists rely on the Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines (1) to initiate or maintain appropriate therapeutic treatments. Recently, some papers have illustrated the need for a standardization of the pre-analytical (2,3) and analytical (4,5) phases. However, to our knowledge, the stability of PTH at −80°C for a long storage period (1 yr or more) has never been studied. To know if there is, or is not, a significant degradation of PTH when samples are stored for a long period, such as in studies where a serum or plasma EDTA bank has been constituted (6–8), might thus be of importance for the conclusions of these studies. The aim of our study was to evaluate the stability of PTH when stored for a long period as serum or plasma EDTA samples at −80°C, and to see if there was an influence of the method (Roche Elecsys or DiaSorin Liaison) on the results obtained.     

Stability of genomic DNA in dried blood spots stored on filter paper

The stability of DNA in dried blood samples obtained from the neonatal screening program in Thailand was retrospectively studied in order to determine the conditions necessary for the long term storage of samples for DNA banking. Specimens from 1991 to 2001, which had been kept in the ambient conditions at the Department of Medical Science, Ministry of Public Health, Thailand, were randomly sampled and used for the study. Genomic DNA was extracted from the samples and DNA fragments of the PAX8 and beta-globin genes were amplified by PCR to determine DNA stability. The study showed that 255-bp and 674-bp fragments of the PAX8 gene could be amplified from all the samples. The DNA fragment of 1,039 bp of the beta-globin gene could be detected in all of the samples for the years 1993 to 2001, but only in seven and five out of the ten studied samples for each of the years 1991 and 1992, respectively. Our study shows that genomic DNA is stable in dried blood stored on filter paper at ambient tropical conditions for at least 11 years. However, DNA quality for amplification of larger DNA fragments decreased when the specimens were stored for longer than 10 years.     

Short report: Rapid DNA extraction from archive blood spots on filter paper for genotyping of Plasmodium falciparum

The practical advantages of sampling and storing blood on filter paper for analyses of human and pathogen genes highlight the need for reliable, sensitive, and cost-effective DNA extraction methods. We describe a new Tris-EDTA (TE) buffer-based method for extraction of DNA from blood dried on filter paper. The method was evaluated against the commonly used methanol and Chelex methods, regarding polymerase chain reaction detection of Plasmodium falciparum parasites from samples stored for 1-2 years. The sensitivity of detection was dependent on the parasite density and type of filter paper. For 3MM Whatman filter paper, the sensitivity was 100%, 73%, and 93% for the TE, methanol, and Chelex methods, respectively. For the longer stored 903 Schleicher & Schuell filter paper, the sensitivity was 93%, 73%, and 0%, respectively. This rapid, simple, and inexpensive extraction method generated superior results from archived specimens compared with the two standard methods and may represent a useful tool in molecular epidemiologic studies.     

Brain natriuretic peptide is stable in whole blood and can be measured using a simple rapid assay: implications for clinical practice

Objectives—To compare the stability of brain natriuretic peptide (BNP) to that of N-terminal atrial natriuretic peptide (NT-ANP) in whole blood and plasma stored under different conditions. To compare a rapid, simple, direct (unextracted) BNP assay to a conventional assay using plasma extraction.
Design—Blinded, prospective, comparative study.
Setting—Tertiary referral cardiology department.
Subjects—Forty two subjects (24 men, 18 women) comprising 28 patients with left ventricular systolic dysfunction (LVSD) ranging from mild to severe and 14 healthy volunteers.
Main outcome measures—Stability of NT-ANP and BNP when stored as whole blood or plasma at room temperature over three days. Reproducibility of measurements.
Results—BNP was stable in whole blood stored at room temperature for three days; mean change in concentration −7.4% (95% CI 0.6 to −14.8), (direct), −6.3% (5.0 to −16.4), (extracted); whereas a significant decline in BNP concentration was noted in plasma stored at room temperature; −23.2% (−13.7 to −31.6), (direct); −14.4% (−3.2 to −24.3), (extracted). By contrast a small non-significant rise in NT-ANP concentration was noted both in whole blood and plasma stored at room temperature for three days; whole blood +8.6% (+22.3 to −3.5), plasma +6.3%, (23.2 to −8.4). The reproducibility of the BNP measurements, and particularly the rapid, direct, measurement, was superior to that for NT-ANP.
Conclusions—BNP is shown to be stable in whole blood for three days and can be measured using a rapid, simple assay. Routine assay of BNP is feasible in ordinary clinical practice and may be of value to general practitioners and hospital based physicians in the diagnosis and management of patients with LVSD. Samples can be sent to a central laboratory without special handling requirements.

Keywords: brain natriuretic peptide;  atrial natriuretic peptide;  heart failure;  diagnosis     

Screening for Human Parvovirus B19 Infection in Egyptian Family Replacement Blood Donors

Up till now, screening for human parvovirus B19 is not routine in national Egyptian blood bank strategy. Blood samples were collected from 500 healthy blood donors within the age range from 18 to 45 years old attending the blood bank of Suez Canal University Hospital, Ismailia, Egypt. Sera were separated and stored at − 20 °C. Serum samples were screened for anti-human parvovirus B19 IgM and IgG antibodies and B19 genome using ELISA and real-time PCR respectively. Frequency of B19 IgM and B19 IgG antibodies was 6.20%, and 80.20% respectively, and the prevalence of B19 genome was 3.00%. There is a high frequency of human parvovirus B19 among Egyptian blood donors; therefore, serological screening for B19 is warranted.     

Analysis on the Effects of External Temperature and Welding Speed on the Safety of EVA Waterproofing Sheet Joints by Hot Air Welding

This study analyzes the optimal seasonal ambient temperature during welding and welding speed conditions for securing high tensile strength of ethylene vinyl acetate (EVA) waterproofing sheets bonded for roofing, installed by hot air welded joints (overlaps). Seven separate ambient temperature conditions (−10, −5, and 0 °C for winter conditions, 20 °C for the normal condition, and 25, 30, and 35 °C for summer conditions) were set for the test variable and seven speed conditions (3, 4, 5, 6, 7, 8, and 9 m/min) for hot air welding. Based on these conditions, EVA sheet joint specimens were prepared, and the tensile strength of the joint sections was tested and measured. Tensile strength results, compared to normal temperature conditions (20 °C) showed an increase in the summer temperature condition but a decrease during winter temperature conditions. The analysis on the effects of the welding speed showed that in summer temperature conditions (25, 30, and 35 °C), the optimum hot air welding speed is 4.3~9.0 m/min at 25 °C, 4.7~8.7 m/min at 30 °C and 5.2~8.6 m/min at 35 °C, whereas in winter (−10, −5, and 0 °C), the optimum hot air welding temperature is 3~4.1 m/min at −10 °C, 3~4.6 m/min at −5 °C and 3~4.9 m/min at 0 °C. Research results demonstrate that it is imperative to consider the welding speed in accordance to the respective seasonal temperature conditions to secure construction quality of the EVA joints for roofing.     

Influence of High Temperature Curing and Surface Humidity on the Tensile Strength of UHPC

The objective of this study is an investigation of the different parameters that influence the tensile strength of ultra-high performance concrete (UHPC). Apart from the shrinkage and stiffness, the tensile strength is an important parameter for the design of crack-free concrete elements, e.g., in machine tool construction. One focus of our work is the influence of concrete curing and the great impact of the mechanical and physical characteristics of hydrated UHPC. For this reason, different curing regimes were investigated. The results show that even after heat treatment or autoclaving, the centric tensile strength of UHPC specimens is strongly influenced by the surrounding ambient humidity. Test specimens that were stored under water after a heat treatment or autoclaving and were still wet during the test had the highest tensile strengths. Storage at 20 °C and 65% relative humidity (rH), however, results in a 25% reduction in tensile strength. Alternating storage between water storage at 20 °C water and storage at 65% rH can also reduce the tensile strength dramatically by up to 70%. In particular, samples that were stored at 65% rH right before testing had very low tensile strengths. Surprisingly, the initially low tensile strength of previously dry stored UHPC can be restored by subsequent water storage. In the absence of any microstructural defects, e.g., microcracks, a possible explanation for this phenomenon can be the stress differences due to a humidity gradient between the core and surfaces and shrinkage combined with a continued reaction of the unhydrated binders of the UHPC.     

High Strain Rate Yielding of Additive Manufacturing Inconel 625 by Selective Laser Melting

Nickel-based alloy Inconel 625, produced by the selective laser melting method, was studied experimentally for its mechanical performance under strain rate loading using Hopkinson bars. Both compression and tensile tests were carried out, with the former also being conducted at 500 °C. The strain rate was in the range of 300 to 3500 s⁻¹ at ambient temperature, and 1200 to 3500 s⁻¹ at the elevated temperature, respectively, for compression tests, and 900 to 2400 s⁻¹ for tensile tests. Results show that the alloy has a strong rate sensitivity with the dynamic yield stress at 3500 s⁻¹, almost doubling the quasistatic value. The test results also show that, even though the temperature elevation leads to material softening, the strain rate effect is still evidential with the dynamic compressive yield stress at the rate 10³ s⁻¹ and 500 °C still being higher than the quasistatic one at ambient temperature. It is also observed that dynamic tensile strengths are generally higher than those of compressive ones at room temperature.     

Osteoblast Attachment Compromised by High and Low Temperature of Titanium and Its Restoration by UV Photofunctionalization

Titanium implants undergo temperature fluctuations during manufacturing, transport, and storage. However, it is unknown how this affects their bioactivity. Herein, we explored how storage (six months, dark conditions) and temperature fluctuations (5–50 °C) affected the bioactivity of titanium implants. Stored and fresh acid-etched titanium disks were exposed to different temperatures for 30 min under wet or dry conditions, and their hydrophilicity/hydrophobicity and bioactivity (using osteoblasts derived from rat bone marrow) were evaluated. Ultraviolet (UV) treatment was evaluated as a method of restoring the bioactivity. The fresh samples were superhydrophilic after holding at 5 or 25 °C under wet or dry conditions, and hydrophilic after holding at 50 °C. In contrast, all the stored samples were hydrophobic. For both fresh and stored samples, exposure to 5 or 50 °C reduced osteoblast attachment compared to holding at 25 °C under both wet and dry conditions. Regression analysis indicated that holding at 31 °C would maximize cell attachment (p < 0.05). After UV treatment, cell attachment was the same or better than that before temperature fluctuations. Overall, titanium surfaces may have lower bioactivity when the temperature fluctuates by ≥20 °C (particularly toward lower temperatures), independent of the hydrophilicity/hydrophobicity. UV treatment was effective in restoring the temperature-compromised bioactivity.     

Correlation between Diarrhea Severity and Oocyst Count via Quantitative PCR or Fluorescence Microscopy in Experimental Cryptosporidiosis in Calves

Cryptosporidium is an important diarrhea-associated pathogen, however the correlation between parasite burden and diarrhea severity remains unclear. We studied this relationship in 10 experimentally infected calves using immunofluorescence microscopy and real-time polymerase chain reaction (qPCR) (N = 124 fecal samples). The qPCR data were corrected for extraction/amplification efficiency and gene copy number to generate parasite counts. The qPCR and microscopic oocyst quantities exhibited significant correlation (R² = 0.33, P < 0.05), however qPCR had increased sensitivity. Upon comparison with diarrhea severity scores (from 0 to 3), a PCR-based count of ≥ 2.6 × 10⁵ parasites or an immunofluorescence microscopy count of ≥ 4.5 × 10⁴ oocysts were discriminatory predictors of moderate-to-severe diarrhea (versus no-to-mild diarrhea), with accuracies and predictive values of 72–82%. In summary, a quantitative approach for Cryptosporidium can refine predictive power for diarrhea and appears useful for distinguishing clinical cryptosporidiosis versus subclinical infection.     

Rapid DNA extraction for molecular epidemiological studies of malaria

DNA isolation from blood samples collected in molecular epidemiological studies is crucial for the quality and reproducibility of data. Blood samples from two malaria endemic sites have been prepared by four different DNA isolation methods with subsequent PCR amplification of the msp2 locus of Plasmodium falciparum. We tested a rapid boiling method; the guanadine isothiocyanate DNA extraction; QIAmp blood kit; and the ISOCODE STIX PCR template preparation dipstick, and analysed the numbers of concurrent infections/sample. The rapid boiling method and the ISOCODE STIX provided overall the best sensitivity combined with ease of handling. The possibility to store and ship the ISOCODE STIX at ambient temperature adds further advantage to this method.     

Dried blood spots,valid screening for viral hepatitis and human immunodeficiency virus in real-life

AIMTo detect chronic hepatitis B (CHB), chronic hepatitis C (CHC) and human immunodeficiency virus (HIV) infections in dried blood spot (DBS) and compare these samples to venous blood sampling in real-life.METHODSWe included prospective patients with known viral infections from drug treatment centers, a prison and outpatient clinics and included blood donors as negative controls. Five drops of finger capillary blood were spotted on filter paper, and a venous blood sample was obtained. The samples were analyzed for HBsAg, anti-HBc, anti-HBs, anti-HCV, and anti-HIV levels as well as subjected to a combined nucleic acid test (NAT) for HBV DNA, HCV RNA and HIV RNA.RESULTSSamples from 404 subjects were screened (85 CHB, 116 CHC, 114 HIV and 99 blood donors). DBS had a sensitivity of > 96% and a specificity of > 98% for the detection of all three infections. NAT testing did not improve sensitivity, but correctly classified 95% of the anti-HCV-positive patients with chronic and past infections. Anti-HBc and anti-HBS showed low sensitivity in DBS (68% and 42%).CONCLUSIONDBS sampling, combined with an automated analysis system, is a feasible screening method to diagnose chronic viral hepatitis and HIV infections outside of the health care system.     

Usefulness of dried blood samples for quantification and molecular characterization of HBV-DNA

The purpose of this study was to assess the use of dried blood spot (DBS) samples for hepatitis B virus (HBV) DNA quantification, HBV genotyping, and detection of G1896A precore mutants and variants in the YMDD polymerase motif. We studied DBS and serum samples from 82 patients with chronic HBV infection (23 hepatitis B e antigen [HBeAg]-positive and 39 HBeAg-negative), 20 HBeAg-inactive carriers, and 15 HBeAg-negative patients under lamivudine therapy (selected from chronic HBV patients). DBS samples consisted of approximately 20 microL of blood applied to 5-mm paper disks. HBV DNA quantification and HBV precore mutant detection were done using real-time polymerase chain reaction, HBV genotyping using restriction fragment length polymorphism, and YMDD variant detection by Inno-lipa assay. DBS and serum results were compared. HBV DNA was detected in a range of 10(2)-10(8) copies/mL, with low intra-assay and inter-assay variation (<10%). Median DBS HBV DNA (copies/mL) was: 3.7 x 10(6) in HBeAg-positive, 6.2 x 10(5) in HBeAg-negative, and 5.5 x 10(2) in inactive carriers (P <.05). HBV DNA was positive in serum (median 5 x 10(3) copies/mL) but negative in DBS for five inactive carriers. The correlation coefficient between HBV DNA concentration in DBS versus serum samples was r(2) = 0.96 (P <.001). The sensitivity of HBV DNA detection in DBS samples was 1 log(10) lower than in serum samples. Concordance between DBS and serum for HBV genotyping, and for precore mutant and YMDD variant detection was optimal. DBS storage for 7 days at room temperature and 21 days at -20 degrees C revealed no decrease in HBV DNA levels or integrity. In conclusion, the DBS sample is useful for HBV DNA quantification, genotyping, and detection of precore mutant and YMDD variants. All four determinations can be completed with a single drop of dried blood.     

Molecular and Serological Characterization of Hepatitis B Virus (HBV)-Positive Samples with Very Low or Undetectable Levels of HBV Surface Antigen

Background: Gaps remain in the detection of nucleic acid test (NAT) yield and occult hepatitis B virus (HBV) infection (OBI) by current HBV surface antigen (HBsAg) assays. The lack of detection may be due to HBsAg levels below current assay detection limits, mutations affecting HBsAg assays or HBsAg levels, or the masking of HBsAg by antibody to HBsAg (anti-HBs). In this study, we evaluate the incremental detection of NAT yield and OBI from five diverse geographic areas by an improved sensitivity HBsAg assay and characterize the samples relative to the viral load, anti-HBs status, and PreS1–S2–S mutations. Included is a comparison population with HBV DNA levels comparable to OBI, but with readily detectable HBsAg (High Surface–Low DNA, HSLD). Methods: A total of 347 samples collected from the USA, South Africa, Spain, Cameroon, Vietnam, and Cote D’Ivoire representing NAT yield (HBsAg(−), antibody to HBV core antigen (anti-HBc)(−), HBV DNA(+), N = 131), OBI (HBsAg(−), anti-HBc(+), HBV DNA(+), N = 188), and HSLD (HBsAg(+), anti-HBc(+), HBV DNA(+), N = 28) were tested with ARCHITECT HBsAg NEXT (HBsAgNx) (sensitivity 0.005 IU/mL). The sequencing of the PreS1–S2–S genes from a subset of 177 samples was performed to determine the genotype and assess amino acid variability, particularly in anti-HBs(+) samples. Results: HBsAgNx detected 44/131 (33.6%) NAT yield and 42/188 (22.3%) OBI samples. Mean HBV DNA levels for NAT yield and OBI samples were lower in HBsAgNx(−) (50.3 and 25.9 IU/mL) than in HBsAgNx(+) samples (384.1 and 139.5 IU/mL). Anti-HBs ≥ 10 mIU/mL was present in 28.6% HBsAgNx(+) and 45.2% HBsAgNx(−) OBI, and in 3.6% HSLD samples. The genotypes were A1, A2, B, C, D, E, F, and H. There was no significant difference between HBsAgNx(−) and HBsAgNx(+) in the proportion of samples harboring substitutions or in the mean number of substitutions per sample in PreS1, PreS2, or S for the NAT yield or OBI (p range: 0.1231 to >0.9999). A total of 21/27 (77.8%) of HBsAgNx(+) OBI carried S escape mutations, insertions, or stop codons. HSLD had more PreS1 and fewer S substitutions compared to both HBsAgNx(−) and HBsAgNx(+) OBI. Mutations/deletions associated with impaired HBsAg secretion were observed in the OBI group. Conclusions: HBsAgNx provides the improved detection of NAT yield and OBI samples. Samples that remain undetected by HBsAgNx have exceptionally low HBsAg levels below the assay detection limit, likely due to low viremia or the suppression of HBsAg expression by host and viral factors.