RT-qPCR Test Research Articles

Rapid spread and association of Schmallenberg virus with ruminant abortions and foetal death in Austria in 2012/2013

Schmallenberg virus (SBV) has emerged in summer–autumn 2011 in north-western Europe. Since then, SBV has been continuously spreading over Europe, including Austria, where antibodies to SBV, as well as SBV genome, were first detected in autumn 2012. This study was performed to demonstrate the dynamics of SBV spread within Austria, after its probable first introduction in summer 2012. True seroprevalence estimates for cattle and small ruminates were calculated to demonstrate temporal and regional differences of infection. Furthermore, the probability of SBV genome detection in foetal tissues of aborted or stillborn cattle and small ruminants as well as in allantoic fluid samples from cows with early foetal losses was retrospectively assessed.SBV first reached Austria most likely in July–August 2012, as indicated by retrospective detection of SBV antibodies and SBV genome in archived samples. From August to October 2012, a rapid increase in seroprevalence to over 98% in cattle and a contemporaneous peak in the detection of SBV genome in foetal tissues and allantoic fluid samples was noted, indicating widespread acute infections. Notably, foetal malformations were absent in RT-qPCR positive foetuses at this time of the epidemic. SBV spread within Austrian cattle reached a plateau phase as early as October 2012, without significant regional differences in SBV seroprevalence (98.4–100%). Estimated true seroprevalences among small ruminates were comparatively lower than in cattle and regionally different (58.3–95.6% in October 2012), potentially indicating an eastward spread of the infection, as well as different infection dynamics between cattle and small ruminants. Additionally, the probability of SBV genome detection over time differed significantly between small ruminant and cattle samples subjected to RT-qPCR testing.

Schmallenberg virus in Dutch dairy herds: Potential risk factors for high within-herd seroprevalence and malformations in calves, and its impact on productivity

In November 2011, the new orthobunyavirus Schmallenberg virus (SBV) was identified in dairy cows that had induced fever, drop in milk production and diarrhoea in the Netherlands (Muskens et al., 2012. Tijdschrift voor Diergeneeskunde 137, 112–115) and a drop in milk production in cows in Northwestern Germany (Hoffmann et al., 2012. Emerging Infectious Diseases 18 (3), 469–472), in August/September 2011. This study aimed at quantifying risk factors for high within-herd prevalence of SBV and SBV-induced malformations in newborn calves in dairy herds in the Netherlands. Additionally, the within-herd impact of SBV infection on mortality rates and milk production was estimated.A case-control design was used, including 75 clinically affected case herds and 74 control herds. Control herds were selected based on absence of malformations in newborn calves and anomalies in reproductive performance. SBV-specific within-herd seroprevalences were estimated. Risk factors for high within-herd SBV seroprevalence (>50%) and the probability of malformed newborn calves in a herd were quantified. In addition, within-herd impact of SBV with regard to milk production and mortality was estimated.Animal-level seroprevalence was 84.4% (95% confidence interval (CI): 70.8–92.3) in case herds and 75.8% (95% CI: 67.5–82.5) in control herds. Control herds that were completely free from SBV were not present in the study. Herds that were grazed in 2011 had an increased odds (OR 9.9; 95% CI: 2.4–41.2)) of a high seroprevalence (>50%) compared to herds that were kept indoors. Also, when grazing was applied in 2011, the odds of malformations in newborn calves tended to be 2.6 times higher compared to herds in which cattle were kept indoors. Incidence of malformations in newborn calves at herd level was associated with both within-herd seroprevalence and clinical expression of the disease in adult cattle.The rate of vertical transmission of SBV to the fetus once a dam gets infected seemed low. A total of 146 stillborn or malformed calves were submitted by 65 farmers during the study period, of which 19 were diagnosed as SBV-positive based on pathological investigation and/or RT-qPCR testing of brain tissue. Based on these results combined with calving data from these herds we roughly estimated that at least 0.5% of the calves born between February and September 2012 have been infected by SBV.A drop in milk production was observed between the end of August 2011 and the first half of September (week 35–36), indicating the acute phase of the epidemic. During a 4-week period in which SBV infection was expected to have occurred, the total loss in milk production in affected dairy herds was around 30–51kg per cow. SBV had no or limited impact on mortality rates which was as expected given the relatively mild expression of SBV in adult cows and the low incidence of malformations in newborn calves.

Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D

A single real-time multiplex quantitative PCR (qPCR) assay for the simultaneous detection of Grapevine virus A, B and D (GVA, GVB and GVD) was developed, using three different fluorescently labeled minor groove binding probes. This multiplex RT-qPCR was compared to singleplex RT-qPCR designed specifically for each virus and a conventional multiplex RT-PCR. The capacity of the multiplex RT-qPCR assay in detecting the three vitiviruses in mixed infections from a range of virus concentrations in the host was assessed. A series of cDNA derived from 48 different grapevine cultivars obtained from diverse geographical regions infected with various isolates and strains of GVA, GVB and GVD were subjected to singleplex, multiplex RT-qPCR, and conventional multiplex RT-PCR testing. The results showed that the developed multiplex RT-qPCR assay was a cost-effective diagnostic tool that could streamline the testing of grapevine vitiviruses, and replace the singleplex RT-qPCR assays, thus reducing time and labor while retaining the same sensitivity and specificity. In particular, no significant differences in detection limits were found between singleplex and multiplex RT-qPCR and specificity was not affected by the inclusion of the three primer/probe combinations within a multiplex RT-qPCR. Comparing the viral load for each virus using singleplex and multiplex RT-qPCR assays revealed no significant differences between the two assays in detecting GVB and GVD. However, while in detecting GVA using singleplex RT-qPCR assay, viral load was higher. Finally, the multiplex RT-qPCR assay was also more sensitive and time efficient than the conventional multiplex RT-PCR that was designed using degenerate primers to detect GVA, GVB and GVD. This multiplex RT-qPCR method could detect viruses in 95.83% of mixed infected samples as compared to 77.08% for multiplex RT-PCR.

Discontinuation of Dasatinib or Nilotinib in Chronic Myeloid Leukemia (CML) Patients (pts) with Stable Undetectable Bcr-Abl Transcripts: Results From the French CML Group (FILMC)

Abstract 604▪FN2▪This icon denotes a clinically relevant abstract Background:Imatinib, the first tyrosine kinase inhibitor (TKI) directed against the Bcr-Abl oncoprotein, has dramatically improved outcomes for pts with CML. Dasatinib and nilotinib, 2 highly potent second generation (2G)-TKI, have been historically licensed for the treatment of pts with resistance or intolerance to imatinib. They have recently received approval in the frontline setting in chronic phase (CP)-CML. Despite the outstanding efficacy of these drugs, their curative potential remains uncertain. Most TKI-treated pts retain residual leukemic cells detected by means of RTQ-PCR and termination of TKI therapy under such circumstances usually leads to disease relapse. Consequently, it is believed that most CML pts require a lifelong TKI treatment. On the contrary, stopping TKI may be envisaged in pts with stable undetectable molecular residual disease (UMRD), as suggested by recent results from the STop IMatinib trial (Mahon et al. Lancet Oncol. 2010), and our observation that dasatinib could be safely stopped in a pt with a stable UMRD suffering from drug-induced pleural effusion (Cony-Makhoul, et al. unpublished). Aims:We asked whether 2G-TKI could be ceased in CML pts with a stable UMRD. The primary objective of our work was to evaluate the risk of loosing major molecular responses (MMR: BCR-ABL/ABL internationally standardized (IS) ratio ≤ 0.1%) by 6 months. Methods:Pts aged at least 18 years with CP-CML and UMRD were proposed dasatinib or nilotinib discontinuation provided that (1) no prior progression to accelerated phase or blast crisis occurred (2) UMRD was sustained on continuing therapy. UMRD was defined by undetectable Bcr-Abl using internationally standardized RTQ-PCR testing performed in local laboratories, providing that at least 20 000 copies of the control gene had been amplified. After 2G-TKI discontinuation, Bcr-Abl transcripts were quantified monthly during the first 6 months and every 2 to 3 months thereafter. Dasatinib or nilotinib were advised to be re-introduced upon loss of MMR. Results:As of August 1, 2011, 25 pts agreed to stop therapy. The results presented here focus on the subgroup of 16 pts with a minimum follow-up (FU) of 6 months (median 15, range: 7–21). These were 9 females and 7 males, with a median age of 59 years (34-81). The Sokal risk group was low in 11/16 (68.75%), intermediate in 2/16 (12.5%), high in 1/16 (6.25%) and unknown in 2/16 (12.5%). Dasatinib (n=9) or nilotinib (n=7) had been administered owing to imatinib grade 2 hematologic or grade 2 to 4 non hematologic intolerance (n=13), secondary imatinib resistance (n=1) or as the frontline drug (n=1). At start of 2G-TKI, 1 pt was in CP, 1 had a complete hematologic response only, 2 had a partial cytogenetic response, 3 had a complete cytogenetic response but lacked MMR, 4 had a MMR with detectable Bcr-Abl transcripts and 5 had a UMRD. The median time on 2G-TKI therapy prior to discontinuation was 32 months (21-56). The median duration of sustained UMRD was 27 months (21-64).Subsequently, MMR was lost in 31.25% (5/16) pts after a median time off-therapy of 4 months (1-5). Treatment was restarted in 4 of these and in an additional pt without MMR loss but showing a detectable MRD on 2 consecutive assessments. Both MMR and UMRD were rapidly regained upon 2G-TKI re-introduction.Eleven pts remained off-therapy at the last follow-up after a median of 13 months (7-20), among which 10 with either a stable UMRD or weakly detectable Bcr-Abl transcripts on one or more occasions. Gender, age, Sokal risk group, type of 2G-TKI, total duration of continuous TKI treatment, duration of 2G-TKI therapy and of UMRD prior to treatment discontinuation did not markedly differ between pts who lost MMR and those with treatment-free persistent MMR but these results may be taken with caution due to the small size of our cohort. Conclusion:2G-TKI may be safely discontinued in CML pts with a long-lasting UMRD under strict molecular monitoring conditions. Importantly, the emergence of a low level of detectable residual disease below the MMR threshold after 2G-TKI withdrawal may not automatically herald CML relapse and may not preclude the possibility to remain treatment-free. A longer follow-up is required to ascertain whether CML will recur. Our study provides a reasonable basis for subsequent large scale prospective trials. Updated results based on a minimal 6 month-FU of the whole cohort will be presented. Disclosures:Rea:Novartis, BMS: Membership on an entity's Board of Directors or advisory committees. Nicolini:Norvartis Pharma France: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb France: Consultancy, Speakers Bureau. Tulliez:Novartis: clinical trial investigator. Guilhot:Novartis, BMS: Membership on an entity's Board of Directors or advisory committees. Mahon:Novartis, BMS: Membership on an entity's Board of Directors or advisory committees.

Abstract 819: Ambient temperature storage of RNA for use in a RT-qPCR test for the detection of GCC mRNA

Abstract The purpose of this study was to evaluate the integrity of T84 cell and lymph node RNA samples, and their use in an RT-qPCR-based diagnostic test, following storage in a chemical matrix designed to stabilize RNA and slow the action of RNase at ambient temperature. Total RNA was purified from the T84 human colonic adenocarcinoma cell line (T84 cells), and from formalin-fixed paraffin-embedded (FFPE) lymph node (LN) tissue. 5µg aliquots of RNA were applied to GenTegra™ RNA tubes, which contain an inert chemical matrix designed to stabilize and protect RNA. RNA samples were stored in the presence or absence of the chemical matrix for 24 hours in the liquid state at 25°C and 37°C, and for two weeks in the dry state at 25°C, 37°C and 56°C. A set of RNA samples was stored at −70°C as a control. Following storage, recovery yields and RNA integrity numbers (RIN) were compared with frozen controls. Transcript-specific cDNA synthesis was performed and the cDNA products were used to conduct qPCR using primers and TaqMan probes targeting β-Actin (ACTB) mRNA for the T84 specimens or using primers and Scorpions probes targeting GCC and ACTB mRNA for the lymph node specimens. T84 cell RNA was incubated in the liquid state at 25°C or 37°C for 24 hours in the presence and absence of a chemical matrix and compared to a control samples stored at −70°C. There was no difference in the RIN between the samples stored at 25°C or 37°C in the presence of the chemical matrix and the frozen control. A decrease in RIN was observed for samples stored at 37°C in the absence of chemical matrix. T84 cell and LN RNA was dried overnight in the presence or absence of a chemical matrix and stored at 25°C, 37°C or 56°C for comparison with a control stored at −70°C. Quantitative recovery was achieved following rehydration of the dried RNA. The RIN of samples stored in the presence of the chemical matrix for two weeks at up to 56°C were not significantly different from control RNA stored at −70°C. Obvious degradation was observed in RNA stored at 25°C, 37°C or 56°C in the absence of chemical matrix. RT-qPCR analysis of the ACTB transcript revealed no difference in Ct values for T84 cell or LN RNA samples stored dry in the presence or absence of chemical matrix and controls stored at −70°. When LN RNA was used for a RT-qPCR diagnostic test for detection of the GCC transcript, the Ct values and copy numbers did not differ significantly between samples stored dry in the presence or absence of a chemical matrix and controls stored at −70°. In this study, we demonstrate that an inert chemical matrix designed to protect against oxidation and hydrolysis and to slow the action of RNase, stabilized RNA samples in the liquid state at 25°C and 37°C for 24 hours, and in the dry state at 25°C, 37°C and 56°C for two weeks. RNA integrity equivalent to frozen controls was maintained during the storage periods, and the chemical matrix had no effect on the results of an RT-qPCR based diagnostic test. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 819.