An integrated disposable device for DNA extraction and helicase dependent amplification

Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium.     

Comparison of eMAG™ versus NucliSENS® EasyMAG® performance on clinical specimens

BackgroundeMAG™ (bioMerieux) is a new nucleic acid extraction platform based on magnetic silica technology, like its predecessor, NucliSENS^® easyMAG^® (bioMerieux). Using the same reagents and disposables, eMAG™ adds further automation, allowing simultaneous extraction of 48 samples directly from primary tubes, and distribution of nucleic acid extracts on PCR strips or in tubes at the end of the extraction process.ObjectiveTo compare the performance of eMAG™ and easyMAG^® on various clinical specimens.Study designRespiratory (n = 199), whole blood (n = 50), plasma (n = 25) and urine (n = 25) specimens were extracted in parallel on both platforms. Both qualitative (respiratory virus, cell control, CMV, EBV, HHV6 and BKV detection) and quantitative (respiratory virus and cell control cycle thresolds, and CMV, EBV, HHV6 and BKV viral loads) results were compared.ResultsDetection of qualitative targets showed good agreement, ranging from 84.6% for whole blood to 95.9% for respiratory specimens. Correlations between quantitative results were good, with R² ranging from 0.802 to 0.995. Quantitative results showed average overall differences below 0.10 log₁₀ copies/mL between eMAG™ and easyMAG^®.ConclusionsThe two platforms showed comparable performance on the types of clinical specimen tested. With higher automation and throughput than easyMAG^®, the eMAG™ platform is likely to be advantageous for laboratories performing a large number of molecular analyses.     

Influence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards

BackgroundInfection with human cytomegalovirus (HCMV) is the most common congenital virus infection, affecting about 0.5–2% of newborns. Using DBS on Guthrie cards, it is possible to discriminate congenital from postnatal HCMV-infection. However, a recent European trial revealed serious problems in detection of low HCMV-DNA levels from DBS-filter-cards (Barbi et al., 2008).⁷ObjectivesEvaluation of the most sensitive combination of sample size, DNA extraction method and PCR system for the detection of low copy numbers of HCMV-DNA from DBS-filter-cards.Study designWe compared three different manual extraction methods for the detection of HCMV-DNA out of DBS: the QIAmp-blood-Mini-Kit, a heat-extraction-method and traditional phenol–chloroform extraction. Additionally, we tested an automated nucleic acid extraction system (NucliSense EasyMag/Biomerieux). Different punch-sizes of DBS spiked with defined HCMV AD169-DNA copy numbers were analyzed. For detection, we used a quantitative in-house-LightCycler-PCR targeting the gB-region using the hybridisation-probe-format. We compared the sensitivity of the real-time-PCR with IE1Ex4-targeted nested-PCR.ResultsThe highest sensitivity with 200 copies HCMV DNA/ml was achieved using the phenol–chloroform method in combination with the nested-PCR and 6 mm, 3 × 3 mm punches or the whole DBS. The QIAmp-blood-Mini-Kit also showed a very high sensitivity by using the whole DBS and the nested-PCR.ConclusionThese results may have strong implications for retrospective diagnosis of congenital HCMV (cHCMV) infection, since a defined combination of the area of punch, the extraction method, and PCR method determine the probability of detection of viral DNA from DBS according to a logistic model.     

Diagnostic performance of near-patient testing for influenza

BackgroundRapid diagnosis of influenza is important for controlling outbreaks and starting antiviral therapy. Direct antigen detection (DAD) is rapid, but lacks sensitivity, whereas nucleic acid amplification testing (NAT) is more sensitive, but also more time-consuming.ObjectivesTo evaluate the performance of a rapid isothermal NAT and two DADs.Study designDuring February–May 2014, we tested 211 consecutive patients with influenza-like illness using a commercial isothermal NAT (Alere™ Influenza A&B) as well as the DAD Sofia^® Influenza A + B and BinaxNOW^® Influenza A&B for detection of influenza-A and -B virus. RespiFinder-22^® a commercial multiplex NAT served as reference test. Serial 10-fold dilutions of influenza-A and -B cell culture supernatants were examined. Another 225 patient samples were tested during December 2014–February 2015.ResultsCompared to RespiFinder-22^®, the isothermal NAT Alere™ Influenza A&B, and the DAD Sofia^® Influenza A + B and BinaxNOW^® Influenza A&B had sensitivities of 77.8%, 59.3% and 29.6%, and specificities of 99.5%, 98.9% and 100%, respectively, for the first 211 patient samples. Alere™ Influenza A&B showed 85.7% sensitivity and 100% specificity in the second cohort. Isothermal NAT was 10-100-fold more sensitive compared to DAD for influenza virus culture supernatants with a lower limit of detection of 5000–50,000 copies/mL. The average turn-around time (TAT) of isothermal NAT and DADs was 15 min, but increased to 110 min for Alere™ Influenza A&B, 30 min for BinaxNOW^® Influenza A&B, and 45 min for Sofia^® Influenza A + B, when analyzing batches of 6 samples.ConclusionSimple sample processing and a TAT of 15 min render isothermal NAT Alere™ Influenza A&B suitable for sequential near-patient testing, but the TAT advantage is lost when testing of larger series.     

Magnetic-silica based nucleic acid extraction for Human Immunodeficiency Virus Type-1 drug-resistance testing in low viremic patients

Human Immunodeficiency Virus Type-1 (HIV-1) drug-resistance testing is challenging for viral loads below 1,000 copies/mL, but, according to HIV-1 guidelines, it should be considered for improving patient management and treatment options. High-recovery and high-purity extraction methods can enhance standard performances of HIV-1 genotyping assays based on direct full-population sequencing.Aim of the present study was to evaluate performances of the NucliSENS easyMAG (NeM) (BioMerieux, Marcy l’Etoile, F) semi-automated nucleic acid extraction system combined with the direct full-population sequencing ViroSeq HIV-1 genotyping (Abbott, IL, US), for detecting drug resistance in samples with HIV-1 RNA < 1,000 copies/mL (n = 62). Data were compared with those from the ViroSeq manual extraction in 86 samples with HIV-1 RNA < 1,000 copies/mL and studied on HIV-1 reference standards.HIV-1 genotyping was successful in 98% of samples extracted with NeM (61/62) and in 84% of those extracted with ViroSeq (72/86) (X² = 8.508, p = 0.004). HIV-1 RNA levels in samples successfully processed with NeM were significantly lower than those in manually processed ones (mean ± SD, respectively, 285 ± 222 copies/mL vs. 403 ± 269 copies/mL) (p = 0.004). For HIV-1 RNA levels <300 and 500 copies/mL, performances of HIV-1 genotyping with NeM were significantly high (97% and 98%, vs. 68% and 78% for manual extraction). As assessed on HIV-1 RNA reference standards, the detection rate at 200 copies/mL for HIV-1 genotyping with NeM extraction was 100%.In conclusion, these data support that HIV-1 direct full-population sequencing combined with NeM is associated with a significantly high success rate, thus improving the management of HIV-1 drug resistance in low viremic patients.     

Automated processing,extraction and detection of herpes simplex virus types 1 and 2: A comparative evaluation of three commercial platforms using clinical specimens

BackgroundRecently, automated platforms have been developed that can perform processing, extraction and testing for herpes simplex virus (HSV) nucleic acid on a single instrument.ObjectivesIn this study, we compared three commercially-available systems; Aptima^®/Panther (Hologic, San Diego, CA), ARIES^® (Luminex Corporation, Austin, TX), and cobas^® 4800 (Roche Molecular Systems Inc, Pleasanton, CA) for the qualitative detection of HSV-1/2 in clinical samples.Study designTwo-hundred seventy-seven specimens (genital [n = 193], dermal [n = 84]) were submitted for routine HSV-1/2 real-time PCR by a laboratory developed test. Following routine testing, samples were also tested by the Aptima, ARIES, and cobas HSV-1/2 assays per the manufacturer’s recommendations. Results were compared to a “consensus standard” defined as the result obtained from ≥3 of the 4 assays.ResultsFollowing testing of 277 specimens, the cobas and ARIES assays demonstrated a sensitivity of 100% for HSV-1 (61/61) and HSV-2 (55/55). The Aptima assays showed a sensitivity of 91.8% (56/61) for HSV-1 and 90.9% (50/55) for HSV-2. Percent specificities for HSV-1 were 96.2% (202/210) by cobas, 99.5% (209/210) by ARIES and 100% (236/236) by Aptima. For HSV-2, the specificities were 98.1% (211/215) by cobas, 99.5% (215/216) by ARIES and 100% (216/216) by Aptima. The turnaround time for testing 24 samples was 2.5 h by the cobas 4800, 3.1 h by Aptima/Panther, and 3.9 h by ARIES.ConclusionsThe three commercial systems can perform all current functions on a single platform, thereby improving workflow and potentially reducing errors associated with manual processing of samples.     

Evaluation of beta-thalassemia in the fetus through cffDNA with multiple polymorphisms as a haplotype in the beta-globin gene

ObjectiveInvasive biopsy during the pregnancy is associated with an abortion risk of approximately 1% for the fetus. Free fetal DNA in maternal plasma is an excellent source of genetic material for prenatal molecular diagnoses. This study was conducted to investigate beta-thalassemia mutation in the fetus through maternal blood with multiple polymorphisms as haplotypes in the beta-globin gene.MethodsIn this study, a total of 33 beta-thalassemia carrier (minor) couples were genotyped by ARMS-PCR for IVSII-IG>A mutation. During pregnancy, 10 mL of blood was collected from pregnant women, and DNA was extracted by the magnetic bead-based extraction, and fetal DNA was enriched with AMPure XP kit. Five polymorphisms in 4 haplotype groups were evaluated by the Sanger Sequencing method. Finally, results were compared with those of the invasion method.ResultsParticipants in study were 33 couples, mean age of the men was 26 ± 5 years, and mean age of women was 23 ± 4 years, and mean MCV, MCH, HbA2 blood parameters were 62.4 ± 5.3, 19.6 ± 3.1, 4.2 ± 2.1 respectively. A total of 33 fetuses were genotyped for IVSII-IG>A mutation. Nine fetuses were affected, 10 fetuses were normal and 14 fetuses were carrier of beta-thalassemia. Sensitivity and specificity of Sanger Sequencing were equal to 88.8% and 91.6% respectively. Positive and negative predictive values were obtained as 80% and 95.6%, respectively.ConclusionMutational status of the fetus can be assessed by determining inheritance of paternally-derived alleles based on detection of haplotype-associated SNP in maternal plasma. Magnetic-based DNA extraction and fetal DNA enrichment are very simple and easy to perform and have satisfactory accuracy.     

The detection,treatment of parvovirus B19 infection induced anemia in solid organ transplants: A case series and literature review of 194 patients

BackgroundThere are no optimal diagnostic, treatment and post-infection surveillance strategies for parvovirus B19 infection in solid organ transplantation (SOT) recipients.MethodsWe conducted a retrospective review of all PVB19 infected cases confirmed by qPCR among SOT recipients at our institution over a 3-year period and reviewed the literature from 1990 to 2021.ResultsEight kidney and two heart transplant patients with refractory anemia had PVB19 infection. The viral DNA load in peripheral blood ranged from 2.62 × 10² to 8.31 × 10⁶ copies/mL. Two patients with the lowest PVB19 DNA load only reduced the use of immunosuppressants and anemia was relieved. Eight received intravenous immunoglobulin (IVIG) (ranging from 0.25 to 0.5 g/kg/day). The median time to anemia improvement (hemoglobulin > 100 g/L) was 16 days (8–70 days) after treatment. One patient had a PVB19 relapse and viral DNA load > 1.00 × 10⁸ copies/mL at diagnosis. A total of 86 studies involving 194 SOTs were screened from the literature, and the most common symptom was anemia and low reticulocyte count. PVB19 DNA was detected in all cases. Of that, 91.4% of cases received IVIG, 53.8% received IVIG and immunosuppression reduction, 6.5% of cases showed reduced immunosuppression without IVIG, and 2.1% did not receive any special treatment. The recurrence rate was 17.5%.ConclusionPVB19 infection is a cause of anemia after SOT, and treatment mainly relies on IVIG and/or immunosuppression reduction.     

Identification of phagocytic cells,NK-like cytotoxic cell activity and the production of cellular exudates in the coelomic cavity of adult zebrafish

Coelomic cavity (CC) cells of mature zebrafish harvested by lavage with media or trypsin–EDTA contained 0.80–1.20 × 10⁵ and 2.0–3.5 × 10⁵ cells, respectively. Media lavage was composed of granulocytes (60–80%), lymphocytes (10–20%), and NCC (4–10%). Granulocytes had large electron dense cytoplasmic paracrystalline granules and a segmented nucleus; they expressed plastin-1, myeloid specific peroxidase and MCSF mRNA; and they were NCAMP-1⁺. Lymphocytes had B- and T-cell specific mRNA and were NCAMP-1^? and NCCRP-1^?. NCC were 3 μm, NCAMP-1⁺ and NCCRP-1⁺ and did not express B- and T-cell specific mRNA. Additionally, trypsin lavage contained monocytes (marginated chromatin, low nuclear:cytoplasm ratio, sparse cytosolic granules) and macrophages (non-segmented nuclei, no margination of chromatin, abundant electron dense granules). E. coli injected into the CC were phagocytosed in a dose and time dependent fashion by granulocytes, monocytes and macrophages. NCC lysed mammalian target cells and NCAMP-1 expressing hybridoma cells in redirected lysis assays.     

Development of a real-time multiplex RSV detection assay for difficult respiratory samples, using ultrasone waves and MNAzyme technology

BackgroundElderly infected with Human Respiratory Syncytial Virus (RSV) often bear low viral loads that stay below the detection limits of commercial assays. A more sensitive detection of RSV infections can improve patient management, guide containment strategies, and possibly prevent morbidity and mortality among populations most severely affected by RSV.ObjectiveTo test the sensitivity for RSV detection by using an alternative extraction method in combination with a new amplification procedure.Study designNasopharyngeal washes and sputum samples (n = 78) form clinical cases, and broncheo-alveolar lavages (n = 27) from an experimental RSV rat model were obtained. An ultrasone-based RNA extraction method was combined with a multi-component Nucleic Acid enzymes (MNAzyme) amplification procedure for simultaneous detection of RSV-A, RSV-B, and an Internal Extraction control IEC.ResultsCompared to standard real-time PCR technology, this method resulted in an increased detection sensitivity, ranging from 0.9 to 4.93 log (average 2.05 ± 1.01) for RSV-A and 0.76 to 4.28 log (average 1.30 ± 0.92) for RSV-B.ConclusionsAn ultrasone-based extraction method with MNAzyme amplification resulted in improved detection of RSV in different respiratory samples, including sputum. This generic method for nucleic acid extraction should be readily applicable for any other respiratory pathogen.     

The mechanical properties of individual,electrospun fibrinogen fibers

We used a combined atomic force microscopic (AFM)/fluorescence microscopic technique to study the mechanical properties of individual, electrospun fibrinogen fibers in aqueous buffer. Fibers (average diameter 208 nm) were suspended over 12 μm-wide grooves in a striated, transparent substrate. The AFM, situated above the sample, was used to laterally stretch the fibers and to measure the applied force. The fluorescence microscope, situated below the sample, was used to visualize the stretching process. The fibers could be stretched to 2.3 times their original length before breaking; the breaking stress was 22 × 10⁶ Pa. We collected incremental stress–strain curves to determine the viscoelastic behavior of these fibers. The total stretch modulus was 17.5 × 10⁶ Pa and the relaxed elastic modulus was 7.2 × 10⁶ Pa. When held at constant strain, electrospun fibrinogen fibers showed a fast and slow stress relaxation time of 3 and 55 s.Our fibers were spun from the typically used 90% 1,1,1,3,3,3-hexafluoro-2-propanol (90-HFP) electrospinning solution and re-suspended in aqueous buffer. Circular dichroism spectra indicate that α-helical content of fibrinogen is ～70% higher in 90-HFP than in aqueous solution.These data are needed to understand the mechanical behavior of electrospun fibrinogen structures. Our technique is also applicable to study other nanoscopic fibers.     

Automated high throughput DNA isolation for detection of human papillomavirus in oral rinse samples

BackgroundOral HPV infection elevates risk of oropharyngeal cancer, but its natural history is unknown. Natural history studies necessitate validation of an automated, high-throughput method for HPV genomic DNA detection in oral rinse samples (ORS).ObjectivesTo compare agreement of oral HPV detection in ORS processed by a magnetic-bead based automated platform to a previous gold-standard, manual protein-precipitation method. Agreement was compared to that of repeat sampling and repeat HPV testing.Study designHIV-infected individuals (n = 100) provided two ORS collected 15 min apart. DNA was isolated from equal aliquots by either a protein-precipitation based (Puregene, Qiagen) or magnetic bead-based (QIAsymphony? SP, Qiagen) method. HPV DNA was detected and type-specified by consensus primer PCR and reverse line blot hybridization. The kappa statistic was used to assess overall agreement (OA) and agreement on a positive test (Ps+).ResultsThe DNA purification methods had very high agreement for categorizing an individual as HPV infected (OA = 0.95; Ps+ = 0.94) as well as for detection of HPV type-specific infection (OA = 0.99; Ps+ = 0.88) in ORS. Agreement for detection of HPV type-specific infection was greater than that observed with repeat oral rinse sampling (OA = 0.99, Ps+ = 0.76) but comparable to inter-assay agreement (OA = 1.00, Ps+ = 0.90).ConclusionsHPV detection in ORS processed with a magnetic-bead based automated platform will facilitate large natural history studies of oral HPV infection necessary to evaluate the potential use of oral HPV detection in oral cancer screening.     

Quantitative PCR of pmoA using a novel reverse primer correlates with potential methane oxidation in Finnish fen

We report a new reverse primer (A621r) for use with A189f in PCR amplification of pmoA alleles in type II methanotrophs. The new primer combination was used to successfully amplify pmoA in peat monolith samples of various depths taken from fen-type peatlands in Finland. In quantitative PCR, pmoA amplicons produced from two sets of three replicate monoliths showed a significant Pearson correlation coefficient (r = 0.77 and 0.61) with methane oxidation potential. The maximum methane oxidation potential and number of pmoA amplicons ranged between 8.8–40.5 μmol g (dry weight)^?1 d^?1 and 5.5 × 10⁷–18.7 × 10⁷ g (wet weight)^?1, respectively, occurring in depths between 10 and 30 cm beneath the surface in the seven individual monoliths used in this study.     

HCV RNA measurement in samples with diverse genotypes using versions 1 and 2 of the Roche COBAS® AmpliPrep/COBAS® TaqMan® HCV test

BackgroundAccurate HCV RNA measurement is required for monitoring treatment. Underquantification has been reported with some genotypes, particularly genotype 4, using version 1 of the Roche COBAS^® AmpliPrep/COBAS^® TaqMan^® HCV Test.ObjectivesCompare the viral loads of clinical specimens representing diverse genotypes from across the United States using versions 1 (V1) and 2 (V2) of the Roche COBAS^® AmpliPrep/COBAS^® TaqMan^® HCV Test. Assess the frequency of nt145 and nt165 variants in the 5′ UTR associated with detection failures and underquantification in a large clinical sample database.Study designThree hundred archived clinical samples were measured using V1 and V2. Bland–Altman analysis was performed on log-transformed results and compared by genotype. The frequencies of nt145 and nt165 variants from 15,817 sequences were calculated.ResultsOn average, V2 results were 0.16 log IU/mL lower than V1 results. The average genotype 4 sample was 0.08 log IU/mL higher in V1 than V2. The largest individual sample differences were −2.10 (genotype 2b) and 1.57 (genotype 2a) log IU/mL. For genotype 4 samples, the greatest underquantification by V1 was 1.46 log IU/mL. There were 13 (0.082%) variants at nt145 and 24 variants at nt165 (0.152%), including one sequence with variants at both positions (0.0063%).ConclusionsGenotype 4 samples from the U.S. are rarely underquantified and not disproportionately so compared to other genotypes using the COBAS^® AmpliPrep/COBAS^® TaqMan^® HCV Tests. Variants at the nt145 and nt165 positions are uncommon in the U.S. and double variants are exceedingly rare. Underquantification of HCV samples with the V1 assay is likely a very rare occurrence in U.S. patients.     

Development of a novel multiplex PCR for the detection and differentiation of Salmonella enterica serovars Typhi and Paratyphi A

In this study, we developed a multiplex polymerase chain reaction (mPCR) assay for pan-Salmonella detection as well as for specific detection of serovars Typhi and Paratyphi A. The assay detects members of the Salmonella genus by amplifying the outer membrane protein C (ompC). The presence of either Salmonella serovar Typhi or Paratyphi A is indicated by amplification of the putative regulatory protein gene STY4220, which is common to both serovars. Further differentiation of the serovars was achieved by targeting the intergenic region (SSPAI) between SSPA1723a and SSPA1724 in serovar Paratyphi A, and stgA, a fimbrial subunit protein, in serovar Typhi. mPCR was evaluated using 124 clinical and reference Salmonella serovars. S. enterica serovars Typhi and Paratyphi A were detected at 100% specificity and sensitivity. Each PCR reaction detected approximately 1 pg of Salmonella genomic DNA. Sensitivity of the PCR when tested on 8-h-enriched spiked blood samples of serovars Typhi and Paratyphi A was estimated at 4.5 × 10⁴–5.5 × 10⁴ CFU/ml, with similar detection levels observed for spiked fecal samples. This mPCR could prove to be a useful diagnostic tool for the detection and differentiation of serovars Typhi and Paratyphi A.     

Une alternative à la chromatographie pour la détection de la macroprolactine : calcul du rapport de deux immunodosages de la prolactine : Immulite 2000® et Kryptor®

Macroprolactin is a high molecular mass form of prolactin with minimal bioactivity in vivo. The presence of macroprolactin must be considered for the differential diagnosis of hyperprolactinemia because prolactin-immunoassays present various reactivity with macroprolactin. So we compared to the reference technique (size-exclusion chromatography) a new screening test: calculation of the ratio between results obtained from two prolactin assays: Immulite^® with high cross reactivity with macroprolactin and Kryptor^® with no reactivity. In this study, serums from 69 patients with various macroprolactinemia (between 8 and 88% with chromatography) were selected and the ratio of the results of two prolactin assays was calculated for 49 hyperprolactinemic serums (prolactinemia > 636 mUI/l, Immulite 2000^®). According to ROC curves analysis, a low Immulite^®/Kryptor^® ratio (< 1.45) indicates the presence of less than 20% of macroprolactin (sensitivity = 100% specificity = 85%), and a high ratio (> 1.80) indicates the presence of more than 50% of macroprolactin (sensitivity = 95%, specificity = 100%). However, this screening test must be confirmed by size-exclusion chromatography for the rare samples presenting an Immulite^®/Kryptor^® ratio between 1.45 and 1.80.     

The role of doxorubicin in non-viral gene transfer in the lung

Proteasome inhibitors have been shown to increase adeno-associated virus (AAV)-mediated transduction in vitro and in vivo. To assess if proteasome inhibitors also increase lipid-mediated gene transfer with relevance to cystic fibrosis (CF), we first assessed the effects of doxorubicin and N-acetyl-l-leucinyl-l-leucinal-l-norleucinal in non-CF (A549) and CF (CFTE29o-) airway epithelial cell lines. CFTE29o- cells did not show a response to Dox or LLnL; however, gene transfer in A549 cells increased in a dose-related fashion (p < 0.05), up to approximately 20-fold respectively at the optimal dose (no treatment: 9.3 × 10⁴ ± 1.5 × 10³, Dox: 1.6 × 10⁶ ± 2.6 × 10⁵, LLnL: 1.9 × 10⁶ ± 3.2 × 10⁵ RLU/mg protein). As Dox is used clinically in cancer chemotherapy we next assessed the effect of this drug on non-viral lung gene transfer in vivo. CF knockout mice were injected intraperitoneally (IP) with Dox (25–100 mg/kg) immediately before nebulisation with plasmid DNA carrying a luciferase reporter gene under the control of a CMV promoter/enhancer (pCIKLux) complexed to the cationic lipid GL67A. Dox also significantly (p < 0.05) increased expression of a plasmid regulated by an elongation factor 1α promoter (hCEFI) approximately 8-fold. Although administration of Dox before lung gene transfer may not be a clinically viable option, understanding how Dox increases lung gene expression may help to shed light on intracellular bottle-necks to gene transfer, and may help to identify other adjuncts that may be more appropriate for use in man.     

Dosage plasmatique des D-dimères sur Integra 800® : comparaison avec le dosage sur Vidas® dans le diagnostic d'exclusion des maladies veineuses thromboemboliques

The D-dimer quantitative test is an exclusive screening tool used by the emergency departments for the exclusion of Deep Venous Thrombosis diseases (DVT). Its negative predictive value is closed to 100%. We compared the diagnostic performance of the D-dimer assay Tinaquant^® performed on an Integra 800^® to the reference Elisa D-dimer assay Vidas^®, which is routinely used in our laboratory as reference test. Interassay reproducibilility (CV) for Tinaquant^® is: 2.34% and 2.05% for levels of 1010 and 4830 ng/ml respectively. We investigated 250 patients with the two tests simultaneously. The correlation obtained through a linear regression (R = 0.86) gives the equation: Y (Integra^®) = 1.11 × (Vidas^®) – 72. The mean D-dimer Vidas^® are: 1337 ± 1700 ng/ml vs. Integra^®: 1496 ng/ml ± 2379. Ninety percent of the results are concordant at a cutoff of 500 ng/ml. Among the 25 discordant results, 18 are Vidas^®(+)/Tinaquant^®(–) showing a better specificity for the Tinaquant^® test, which is confirmed by imaging results for DVT diagnosis. Only 7 of the 25 give a better specificity for Vidas^®. Tinaquant^® D-dimer assay demonstrated interesting performances in this study regarding the practicability and the efficiency of the test. It appears to be suitable for the exclusion of DVT diseases and should be studied throughout a larger number of outpatients.