PCR detection of dengue virus using dried whole blood spotted on filter paper

Whole blood dried onto filter paper constitutes a potentially useful material for molecular testing of viruses, including dengue. In order to assess the stability of viral RNA, we carried out dengue-RNA detection in whole blood infected with dengue virus that had been previously spotted onto filter paper. Filter papers were stored at room temperature, 4 and -70 degrees C and processed for PCR assay at intervals of 2, 4, 6 and 9 weeks. Our results demonstrated that dengue-RNA was stable in filter paper for 9 weeks at all tested temperatures. Furthermore, we evaluated these conditions using frozen sera and dried blood samples onto filter paper from 52 patients with confirmed clinical diagnosis of dengue infection. PCR results showed a 100% specificity and 93% sensitivity for dried blood samples. This storage method facilitates the transportation and analysis by nucleic acid amplification techniques even when freezing conditions are not available.     

Comparison of manual and automated DNA purification for measuring TREC in dried blood spot (DBS) samples with qPCR

Automated nucleic acid extractions from dried blood spot (DBS) samples promises standardized sample treatment, low error rates, avoidance of contamination and requirement of less hands-on time. In the present study, non-automated and automated column based extraction processes using the QIAamp Investigator procedure were compared for the extraction of DNA from DBS samples. The concentration and the purity of DNA generated were determined by optical density readings. Furthermore qPCR downstream applications using the nucleic acids extracted with the two processes and albumin and T-cell receptor excision circles (TREC) copy numbers were measured and compared. The influence of the time of storage was also investigated by analyzing samples freshly dried and stored up to 11weeks at -20°C from the same individual. Finally, we provide arguments of preferentially choosing the automated procedure for extracting DNAs from DBS samples when downstream qPCR applications are required.     

Phenylalanine and tyrosine measurements across gestation by tandem mass spectrometer on dried blood spot cards from normal pregnant women

PurposeMaternal phenylketonuria (MPKU) requires strict control of phenylalanine (Phe) and supplemental tyrosine (Tyr). Monitoring during pregnancy using dried blood spot (DBS) cards by tandem mass spectrometry (MS/MS) is now standard practice, however there are no Phe and Tyr reference ranges for DBS MS/MS method in healthy pregnant women.MethodsDBS cards (63–1364 days in storage) from healthy women with singleton pregnancies were analyzed by MS/MS. Three hundred ninety DBS cards from 170 pregnancies (5/1–39/6 weeks’ gestation), were tested.ResultsBoth Phe and Tyr levels declined from the first trimester (Phe: 36.2 +/− 10.6; Tyr 25.7 +/− 9.7 µmol/L) to the second trimester (Phe 33.4+/−9.3; Tyr 21.7+/− 6.7 µmol/L) and remained stable in the third trimester (Phe 32.3 +/− 8.7; Tyr 21.0 +/− 6.6 µmol/L). Phe and Tyr levels declined over time since collection (Phe: 0.004 µmol/L per day; Tyr 0.002 µmol/L). Nomograms by gestational age were created using raw data and data adjusted for time from sample collection. Reference ranges by trimester are provided.ConclusionsBoth Phe and Tyr decline quickly during the first trimester and remain relatively constant over the second and third trimesters. These nomograms will provide a valuable resource for care of MPKU.     

Use of whole blood dried on filter paper for detection and genotyping of measles virus

PCR, and two different ELISAs were used to detect measles virus on blood dried on filter paper samples from Equatorial Guinea. Sensitivity was 40% by PCR, 57.1% by indirect ELISA and 86.7% by micro chain capture ELISA. Genotype B3 was found in two positive samples by PCR.     

Comparison of the performance of enzyme immunoassays for hepatitis B and C detection in dried blood spot

Dried blood spots (DBSs) could be an alternative to serum for hepatitis B virus (HBV) and hepatitis C virus (HCV) diagnosis. This study aims to evaluate two enzyme immunoassays (EIAs) for HBsAg and anti-HCV detection using DBS. Serum was tested using commercial EIA. DBS was tested using optimized EIA developed for serum and commercial EIA developed for DBS (Imunoscreen). Concordances between DBS and serum samples for both markers and EIAs were higher than 97%. Both EIAs demonstrated good performance for HBsAg and anti-HCV detection using DBS, and these methods could be used unchangeably increasing the access for HBV and HCV diagnosis.     

荧光测定法定量测定滤纸片干血斑标本G6PD酶活性的可靠性分析

目的探讨荧光测定法测定滤纸片干血斑标本G6PD酶活性的可靠性。方法随机选取43例门诊孕前咨询者为检测对象,每人采集2ml静脉血抗凝保存同时制备滤纸片干血斑标本。采用G6PD/6PGD比值法测定抗凝血G6PD/6PGD比值,同时采用荧光测定法定量测定滤纸片干血斑标本G6PD酶活性,比较两种方法测定结果。结果荧光测定法检测43份滤纸片干血斑标本,检出1例缺乏（1.57 IU/gHb）;比值法检测43份抗凝血标本,检出1例缺乏（0.76）;两种方法符合率为100%。结论荧光测定法定量测定G6PD酶活性准确性高、简单、快捷、费用低廉,可对滤纸片干血斑标本进行G6PD缺乏症的大规模筛查,适于在G6PD缺乏高发区推广应用。     

Comparison of two nucleic acid extraction and testing systems for HCMV-DNA detection and quantitation on whole blood specimens from transplant patients

Quantitative detection of human cytomegalovirus (HCMV) DNA on whole blood is currently the primary choice for virological monitoring in transplant patients and for determining the appropriate antiviral strategy, however specific issues of variability remain in terms of extraction methods, amplification efficiency, and variability. This study compared the performance characteristics of two nucleic acid extraction and testing systems for HCMV-DNA quantitation, the artus^® CMV QS-RGQ kit, associated with a fully automated DNA extraction and assay set up by Qiagen (system 1) and the Q-CMV Real Time Complete kit by Nanogen, associated with a semiautomated nucleic acid extraction system by Biomérieux (system 2) in 189 specimens from transplant patients and 10 from 2012 HCMV Quality Control for Molecular Diagnostics (QCMD). The two systems exhibited a 80.4% concordance. Differences between the two systems were within ±1 log₁₀ copies/ml of the averaged log₁₀ results for 88.9% of the tested specimens. For all qualitatively discordant specimens, mean viral load was ≤3 log₁₀ copies/ml. Considering viral load measurement, system 1 gave earlier positives that system 2, with a 14.8% of specimens resulted positive at low viral loads with system 1 and negative with system 2. In QCMD specimens, difference was below 0.7 log₁₀ copies/ml for both the systems.     

Optimized PCR assay for detection of white spot syndrome virus (WSSV)

A rapid PCR assay for detection of white spot syndrome virus (WSSV) was developed based on the nested PCR procedure described by Lo et al. (1996) and outlined as the recommended PCR diagnostic assay in the Manual of Diagnostic Tests for Aquatic Animals published by the Office of International Epizootics (OIE, 2009). The optimized procedure incorporated the second step primers used in the nested WSSV PCR. By adjusting the annealing temperature and shortening the cycling times, this modified assay is substantially faster and as sensitive as the recommended OIE protocol. The modified PCR test was compared directly to the two-step nested PCR protocol and a modified nested procedure. The sensitivity of the published assay was determined by template dilutions of semi-purified WSSV virions that had been quantitated using real-time PCR for detection of WSSV. Various isolates were tested using the modified procedure, to ensure that the assay was able to detect WSSV from different geographical locations.     

Evaluation of DNA extraction methods for dried blood spots in the diagnosis of congenital cytomegalovirus infection

BackgroundDried blood spots (DBS) may be valuable in the diagnosis of congenital cytomegalovirus (CMV) infection. However, the 2007 European Quality Control for Molecular Diagnostics (QCMD) proficiency testing programme showed that CMV DNA detection in DBS was lacking sensitivity in a considerable number of participating laboratories.ObjectiveTo compare DNA extraction methods for DBS for detecting CMV. Sensitivity and applicability of the methods for high-throughput usage were assessed.Study designGuthrie cards were spotted with CMV DNA-positive whole blood (n = 15). DNA was extracted from the DBS using different extraction methods, followed by CMV amplification by means of real-time PCR.ResultsSignificant differences between the extraction methods with respect to the sensitivity were found. Optimal sensitivity was achieved when samples were tested in triplicate, demonstrating that the methods in general operated around their detection limits. Triplicate testing using the protocol by Barbi et al. [Barbi M, et al. Cytomegalovirus DNA detection in Guthrie cards: a powerful tool for diagnosing congenital infection. J Clin Virol 2000;17:159–65], representing the most sensitive methods, resulted in sensitivities of 100%, 86%, and 50% for DBS with CMV DNA loads of 5–4, 4–3, and 3–2 log₁₀ copies/ml, respectively. This indicates that sensitivity limitations apply in the clinically relevant concentration range. Few methods appeared suitable for 96-well format high-throughput testing.DiscussionWhen considering universal neonatal screening for congenital CMV infection, an assay which is both sensitive and applicable for high-throughput testing is required. The protocol by Barbi et al. and the BioRobot Universal System appear appropriate candidates currently available for 96-well format application in neonatal screening using DBS.     

Use of dried blood on filter paper in the ELISA for Brucella abortus

An ELISA for Brucella abortus antibody detection using blood collected on filter paper is described. The method gave similar results to the complement fixation test. The signal-noise ratio was good. The system offers considerable advantages when transport of serum samples to the laboratory causes problems.     

Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens

An evaluation of the sensitivities of three DNA extraction methods, i.e., FTA filter paper, a QIAamp stool mini kit, and a conventional phenol-chloroform method, by using specimens with known concentrations of Enterocytozoon bieneusi spores was performed. FTA filter paper and the QIAamp stool mini kit were the most sensitive methods, which could detect E. bieneusi in specimens with a concentration of 800 spores/ml. We also compared five previously described PCR methods that use five different primer pairs for the detection of E. bieneusi and showed that MSP3-MSP4B and EBIEF1-EBIER1 were the most sensitive primers. Although both sets of primers showed the same sensitivity, using the MSP3-MSP4B primers can directly provide genotypic information by sequencing. A blinded diagnostic test to compare PCR and light microscopy methods for the detection of E. bieneusi in stool specimens was also conducted. The use of FTA filter paper for DNA extraction together with the PCR method using the primer pair MSP3-MSP4B showed 100% sensitivity and 100% specificity for the detection of E. bieneusi in stool specimens, while the light microscopy method gave a sensitivity of 86.7% and a specificity of 100%.     

Comparison of different methods of bacterial detection in blood components

Background Over the last two decades, the residual risk of acquiring a transfusion-transmitted viral infection has been reduced to less than 1 : 1 000 000 via improvements in different techniques (e.g. donor selection, leuco-depletion, introduction of 3rd or 4th generation enzyme-linked immunosorbent assays and mini-pool nucleic acid testing (MP-NAT). In contrast, the risk for transfusion-associated bacterial infections has remained fairly stable, and is estimated to be in a range between 1 : 2000 and 1 : 3000. Platelets are at an especially higher risk for bacterial contamination, because they are stored at room temperature, which provides good culture conditions for a broad range of bacterial strains. To improve bacterial safety of blood products, different detection systems have been developed that can be divided into culture systems like BacT/ALERT or Pall eBDS, rapid detection systems like NAT systems, immunoassays and systems based on the FACS technique. Culture systems are used for routine bacterial screening of platelets in many countries, whereas rapid detection systems so far are mainly used in experimental spiking studies. Nevertheless, pathogen-reduction systems are currently available for platelet concentrates and plasma, and are under investigation for erythrocytes. Methods In this review, the functional principles of the different assays are described and discussed with regard to their analytical sensitivity, analytical specificity, diagnostic sensitivity, diagnostic specificity and clinical efficiency. The detection methods were clustered into three groups: (i) detection systems currently used for routine screening of blood products, (ii) experimental detection systems ready to use for routine screening of blood products, and (iii) new experimental detection systems that need to be investigated in additional spiking studies and clinical trials. Results A recent International Society of Blood Transfusion international forum reported on bacterial detection methods in 12 countries. Eight countries have implemented BacT/ALERT into blood donor screening, whereas in three countries only quality controls were done by culture methods. In one country, shelf-life was reduced to 3 days, so no bacterial screening was implemented. Screening data with culture methods can be used to investigate the prevalence of bacterial contamination in platelets. Differing results between the countries could be explained by different test definitions and different test strategies. Nevertheless, false-negative results causing severe transfusion-related septic reactions have been reported all over the world due to a residual risk of sample errors. Rapid screening systems NAT and FACS assays have improved over the last few years and are now ready to be implemented in routine screening. Non-specific amplification in NAT can be prevented by pre-treatment with Sau3AI, filtration of NAT reagents, or reduction of the number of polymerase chain reaction cycles. FACS systems offer easy fully automated handling and a handling time of only 5 min, which could be an option for re-testing day-5 platelets. New screening approaches like immunoassays, detection of bacterial adenosine triphosphate, or detection of esterase activity need to be investigated in additional studies. Conclusion Bacterial screening of blood products, especially platelets, can be done with a broad range of technologies. The ideal system should be able to detect one colony-forming unit per blood bag without a delay in the release process. Currently, we are far away from such an ideal screening system. Nevertheless, pathogen-inactivation systems are available, but a system for all blood components will not be expected in the next few years. Therefore, existing culture systems should be complemented by rapid systems like NAT or FACS especially for day-5 platelets.     

Use of dried blood spot specimens in the detection of human immunodeficiency virus type 1 by the polymerase chain reaction.

Dried blood spots (DBSs) constitute a potentially valuable source of material for human immunodeficiency virus (HIV) serologic and molecular testing. To facilitate molecular testing, we have adapted the polymerase chain reaction (PCR) to the detection of HIV proviral DNA in DBS samples. The method is highly reproducible, with 75 microliters of whole dried blood providing sufficient DNA for duplicate testing with three primer sets. By using DBS PCR, 66 of 69 (95.6%) seropositive at-risk individuals tested positive by at least two primer sets and 85 of 85 (100%) low-risk seronegative blood donors tested negative by all three sets of primers. The frequency of HIV DNA detection in seronegative at-risk individuals was low, with only 1 of 58 (1.7%) individuals testing positive. These results show that in a clinical environment, HIV PCR analysis of DBS specimens is specific and sensitive. The method is cost effective and presents a useful alternative to the isolation of HIV from seropositive babies with an undefined infection status.     

Immunoreactive Trypsinogen (IRT) as a Biomarker for Cystic Fibrosis: challenges in newborn dried blood spot screening

On May 23-24, 2011, a workshop entitled "Immunoreactive Trypsinogen (IRT) as a Biomarker for Cystic Fibrosis: Technical Issues and Challenges" was held in Annapolis, Maryland. The two-day workshop was co-hosted by the National Newborn Screening and Genetics Resource Center, Austin, Texas, and the Association of Public Health Laboratories, Silver Spring, Maryland, in collaboration with the Health Resources and Services Administration and the Centers for Disease Control and Prevention. Participants included nearly 40 representatives from U.S. state public health and commercial laboratories performing newborn dried blood spot screening tests for cystic fibrosis (CF), the federal government, academic research institutions, and commercial vendors of products used in newborn screening. Representatives from selected European CF newborn screening programs were also present. The workshop focused on identifying key IRT testing issues and mechanisms for achieving their resolution and laboratory harmonization in order to reduce, or eliminate completely, the late identified CF cases following a negative newborn screen. Informative findings are reported, their impacts on improving IRT screening are described, and their implications are discussed.     

Single tube multiplex PCR detection of 27 cystic fibrosis mutations and 4 polymorphisms using neonatal blood samples collected on Guthrie cards

In response to recommendations for cystic fibrosis (CF) carrier screening of the American College of Medical Genetics/American College of Obstetrics and Gynecology (ACMG/ACOG), we evaluated a commercial kit for mutation panel testing (Roche CF Gold Linear Array Panel). This kit tests for 25 CF mutations and 4 polymorphisms; it comprises an analyte specific reagent for single tube multiplex polymerase chain reaction (PCR) amplification and subsequent allele specific oligonucleotide (ASO) hybridization. Neonatal whole blood samples collected on Guthrie card filter paper served as the DNA source. Following a wash step to remove whole blood PCR inhibitors, multiplex PCR amplification could be performed either on DNA that was heat extracted from Guthrie cards or directly on the filter paper matrix itself. In 13 CF patient samples, the CF mutation results obtained with this kit agreed completely with those obtained from a reference laboratory that performs an 87 CF mutation panel. The kit was reliable, despite the small sample size (3 mm diameter punch of the Guthrie card), the presence of PCR inhibitors in whole blood, and protracted storage of blood samples (up to 9 yr at room temperature). The kit was convenient, cost competitive, and practical for use in a small CF screening laboratory.     

Evaluation of the sensitivities of DNA extraction and PCR methods for detection of Giardia duodenalis in stool specimens

Sensitivities of DNA extraction methods and PCR methods for Giardia duodenalis were evaluated. A combination of the most sensitive methods, i.e., FTA filter paper and a PCR protocol using RH11/RH4 and GiarF/GiarR primers, showed no significant differences compared to immunofluorescence assay in terms of their sensitivities and specificities.