Flinders Technology Associates Cards Research Articles

Evaluation of Flinders Technology Associates cards for storage and molecular detection of avian metapneumoviruses

The feasibility of using Flinders Technology Associates (FTA) cards for the molecular detection of avian metapneumovirus (aMPV) by reverse transcriptase-polymerase chain reaction (RT-PCR) was investigated. Findings showed that no virus isolation was possible from aMPV-inoculated FTA cards, confirming viral inactivation upon contact with the cards. The detection limits of aMPV from the FTA card and tracheal organ culture medium were 101.5 median ciliostatic doses/ml and 100.75 median ciliostatic doses/ml respectively. It was possible to perform molecular characterization of both subtypes A and B aMPV using inoculated FTA cards stored for up to 60 days at 4 to 6°C. Tissues of the turbinate, trachea and lung of aMPV-infected chicks sampled either by direct impression smears or by inoculation of the tissue homogenate supernatants onto the FTA cards were positive by RT-PCR. However, the latter yielded more detections. FTA cards are suitable for collecting and transporting aMPV-positive samples, providing a reliable and hazard-free source of RNA for molecular characterization.

Analysis of class I and II aberrations in Iraqi childhood acute myeloid leukemia using filter paper cards

The lack of molecular diagnosis in the field of cancer in Iraq has motivated us to perform a genetic analysis of pediatric acute myelogenous leukemia (AML), including class I and II aberrations. Peripheral blood or bone marrow cells were collected from 134 AML children aged ≤15years. Flinders Technology Associates (FTA) filter paper cards were used to transfer dried blood samples from five Iraqi hospitals to Japan. DNA sequencing was performed to identify class I mutations. Nested RT-PCR was used to detect class II aberrations, except that MLL rearrangement was detected according to long distance inverse-PCR. NPM1 and FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations were analyzed by GeneScan using DNA template. Among 134 Iraqi pediatric AML samples, the most prevalent FAB subtype was M2 (33.6%) followed by M3 (17.9%). Class I mutations: 20 (14.9%), 8 (6.0%), and 8 (6.0%) patients had FLT3-ITD, FLT3-TKD, and KIT mutations, respectively. Class II mutations: 24 (17.9%), 19 (14.2%), and 9 (6.7%) children had PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 transcripts, respectively. MLL rearrangements were detected in 25 (18.7%) patients. NPM1 mutation was detected in seven (5.2%) cases. Collectively, approximately 30% of AML children were proved to carry favorable prognostic genetic abnormalities, whereas approximately 10% had high FLT3-ITD allelic burden and needed a special treatment plan including allogeneic hematopoietic stem cell transplantation. Acute promyelocytic leukemia (APL) was frequent among Iraqi pediatric AML. It is likely that molecular diagnosis using FTA cards in underdeveloped countries could guide doctors towards an appropriate treatment strategy.

Evaluation of FTA cards as a sample collection method for detection of BVDV in persistently infected beef cattle

The accuracy of test outcome is directly related to the quality of samples arriving at the diagnostic laboratory. However, in certain field situations sample storage conditions may be less than optimal. Flinders Technology Associates (FTA) cards are a sample collection and storage tool suitable for field conditions. For the evaluation of FTA cards, nasal swabs and EDTA blood samples were collected by traditional methods from a group of known bovine viral diarrhea virus (BVDV) persistently infected (PI) animals, as well as from animals in a herd never diagnosed with BVDV infection. Diagnostic performance parameters of the reverse transcription real-time PCR assay (RT-r PCR) were compared between whole-blood and nasal-swab samples collected onto FTA cards from the same animals taken at the same time. Sensitivity and specificity based on the dichotomous classification of BVDV-positive or negative were 100% for the samples collected using traditional methods, and for those collected onto FTA cards. Real-time PCR cycle threshold values demonstrated a lower analytic sensitivity for samples collected onto FTA cards, although samples from FTA cards correctly identified all PI animals tested. One animal from the PI group tested BVDV-positive only in the nasal specimen, suggesting the positive resulted from contact exposure rather than from systemic viral production. To further evaluate the potential of the FTA cards, a subset of BVDV-positive samples (n=29) were sub-typed by direct sequencing of the BVDV qRT PCR products obtained from the case material. BVDV-1 was detected in 75.9% (22/29) of cases, BVDV-2 in 13.8% (4/29) of cases, and both serotypes were simultaneously detected in the remaining 10.3% (3/29) cases. Time savings associated with use of FTA cards for sample collection was calculated to be 23.5 seconds per blood sample. The FTA cards provide a method for diagnostic sample handling that helps overcome sample quality issues associated with improper storage conditions and shipping concerns.

A Simple Non-Powered Passive Trap for the Collection of Mosquitoes for Arbovirus Surveillance

Mosquitoes often are collected as part of an arbovirus surveillance program. However, trapping and processing of mosquitoes for arbovirus detection is often costly and difficult in remote areas. Most traps, such as the gold standard Center for Disease control light trap, require batteries that must be charged and changed overnight. To overcome this issue we have developed several passive traps for collection of mosquitoes that have no power requirements. The passive traps capture mosquitoes as they follow a CO2 plume up a polyvinyl chloride pipe leading to a clear chamber consisting of a plastic crate. We believe the translucent, clear windows created by the crate inhibits escape. Once inside the crate mosquitoes readily feed on honey-treated Flinders Technology Associates cards that then can be processed by polymerase chain reaction for viral ribonucleic acid. Of the two designs tested, the box or crate-based passive trap (passive box trap, PBT) generally caught more mosquitoes than the cylinder trap. In Latin square field trials in Cairns and Florida, PBTs collected mosquitoes at rates of 50 to 200% of Center for Disease Control model 512 light traps. Mosquito collections by PBTs can be increased by splitting the CO2 gas line so it services two traps, or by placing an octenol lure to the outside of the box. Very large collections can lead to crowding at honey-treated cards, reducing feeding rates. Addition of fipronil to the honey killed mosquitoes and did not impact feeding rates nor the ability to detect Kunjin viral ribonucleic acid by polymerase chain reaction; this could be used to minimize crowding affects on feeding caused by large collections. The passive traps we developed are made from inexpensive, commonly available materials. Passive traps may thus be suitable for collection of mosquitoes and potentially other hematophagous dipterans for pathogen surveillance.

A Comparison of Two Methods of Eluting Insect Dna from Flinders Technology Associates Cards

Flinders Technology Associates (FTATM) cards contain chemicals that lyse cells and protect DNA from degradation for room-temperature storage (WhatmanTM, 2009). Consequently, DNA extrac tion takes less time and storage requires less en ergy and space than traditional methods requir ing lysis, purification, and -20 °C storage in tubes. These cards are also easy to transport and face fewer shipping restrictions and less opportunity for damage than mounted specimens or those stored in alcohol. DNA stored on FTATM cards pro duces a full allele compliment during genotyping after 11 yr (Kline, 2010) and the manufacturer re ports successful PCR with FTATM stored DNA for 17 yr and counting (WhatmanTM, 2009). Despite these benefits, this technology has been utilized infrequently with insects and other arthropods (Miller et al. 2012). Desloire et al. (2006) report ed mixed results obtaining successful PCR from mite DNA and Bujang et al. (2011) were unable to obtain successful PCR reactions from termite DNA when using the manufacturer's protocol. This lead to the development of an alternative method of preparing an FTATMarchived sample for successful PCR (Bujang et al. 2011). There may be several hurdles impeding broader use of the FTATM technology by arthropod research ers. First, the DNA quality and quantity of the washed, single-use, FTATM disc is untestable leading to uncertainty about the cause when PCR fails and second, the wash reagents are relatively expensive ($0.23-$0.36 for two 200uL FTATM puri fication reagent washes). Initial attempts to PCR amplify FTATM archived whitefly DNA using the manufacturer's protocol and the methods of Bu jang et al. (2011) yielded mixed results but PCR was successful when discs were boiled for 5 min in

Evaluation of Flinders Technology Associates cards for collection and transport of samples for detection of Porcine reproductive and respiratory syndrome virus by reverse transcription polymerase chain reaction

Blood, tissue and oral fluid samples collected from experimentally infected animals and field cases were used to evaluate the safety, diagnostic sensitivity and specificity of Flinders Technology Associates (FTA) cards for Porcine reproductive and respiratory syndrome virus (PRRSV) reverse transcription polymerase chain reaction (RT-PCR) diagnostics. The analytical sensitivity of PRRSV RT-PCR from serum and oral fluids in FTA cards was reduced, although the virus could still be detected at concentrations of 10(1) and 10(3) TCID/ml, respectively. The sensitivity and specificity of PRRSV RT-PCR detection from serum, blood, and tissue samples in cards collected from experimentally infected animals were 100%. Sensitivity for oral fluids was 45% (95% CI: 19.97-73.01) compared to fresh. For field samples, sensitivity was 89% (95% CI: 77.35-95.63) and 100% (95% CI: 80.00-100) for serum and lung samples, respectively. The sensitivity was the same for samples stored in cards at room temperature or at 4ºC, and tested overnight or after 14 days. Cards inoculated with PRRSV-positive samples did not yield replicating virus after cell culture. In conclusion, FTA cards proved to be a safe, simple, and sensitive alternative method to transport serum, blood, and tissue samples for PRRSV RT-PCR diagnostics; however, a significant decrease in RT-PCR sensitivity should be expected from oral fluid samples.

Comparative Detection of Trypanosomal DNA by Loop-Mediated Isothermal Amplification and PCR from Flinders Technology Associates Cards Spotted with Patient Blood

We analyzed DNA eluted from FTA (Flinders Technology Associates) cards spotted with blood from human African trypanosomiasis (HAT) patients admitted at Lwala Hospital in eastern Uganda and Kaliua Health Centre in northwestern Tanzania. The aims were to evaluate loop-mediated isothermal amplification (LAMP) for detection of trypanosomal DNA in clinical samples and to characterize the infecting trypanosomes to the subspecies level. LAMP targeting the Trypanozoon conserved random inserted mobile element (RIME-LAMP) and that for the serum resistance-associated (SRA) gene (SRA-LAMP) were performed. For comparison, PCRs for the SRA gene specific for Trypanosoma brucei rhodesiense (SRA-PCR) and that to amplify the Trypanosoma brucei gambiense-specific surface glycoprotein (TgSGP-PCR) were done. Out of 128 samples analyzed, SRA-PCR was positive in 101 samples (78.9% sensitivity; 95% confidence interval [CI], 71.1 to 85.1%), SRA-LAMP was positive in 120 (93.8%; 95% CI, 88.2 to 96.8%), while RIME-LAMP revealed signals in 122 (95.3%; 95% CI, 90.2 to 97.8%). RIME-LAMP and SRA-LAMP were each significantly more sensitive than SRA-PCR (P values of 0.000 and 0.001, respectively; Fisher's exact test). There was poor agreement between RIME-LAMP and SRA-LAMP and the SRA-PCR, yielding kappa values of 0.31 and 0.40, respectively. Agreement between SRA-LAMP and RIME-LAMP was almost perfect (kappa value, 0.85; 95% CI, 0.64 to 1). All the 128 field samples were negative by TgSGP-PCR. Blood spots from three T. b. gambiense HAT cases from northwestern Uganda were positive by TgSGP-PCR and RIME-LAMP. PCR took five times longer to execute than LAMP. LAMP may be useful to monitor emerging HAT foci or to test travelers returning from countries where HAT is endemic. It should be evaluated in a case-control study to determine its utility as a HAT diagnostic.

Validation of Marek's Disease Diagnosis and Monitoring of Marek's Disease Vaccines from Samples Collected in FTA® Cards

The use of Flinders Technology Associates (FTA) filter cards to quantify Marek's disease virus (MDV) DNA for the diagnosis of Marek's disease (MD) and to monitor MD vaccines was evaluated. Samples of blood (43), solid tumors (14), and feather pulp (FP; 36) collected fresh and in FTA cards were analyzed. MDV DNA load was quantified by real-time PCR. Threshold cycle (Ct) ratios were calculated for each sample by dividing the Ct value of the internal control gene (glyceraldehyde-3-phosphate dehydrogenase) by the Ct value of the MDV gene. Statistically significant correlation (P < 0.05) within Ct ratios was detected between samples collected fresh and in FTA cards by using Pearson's correlation test. Load of serotype 1 MDV DNA was quantified in 24 FP, 14 solid tumor, and 43 blood samples. There was a statistically significant correlation between FP (r = 0.95), solid tumor (r = 0.94), and blood (r = 0.9) samples collected fresh and in FTA cards. Load of serotype 2 MDV DNA was quantified in 17 FP samples, and the correlation between samples collected fresh and in FTA cards was also statistically significant (Pearson's coefficient, r = 0.96); load of serotype 3 MDV DNA was quantified in 36 FP samples, and correlation between samples taken fresh and in FTA cards was also statistically significant (r = 0.84). MDV DNA samples extracted 3 days (t0) and 8 months after collection (t1) were used to evaluate the stability of MDV DNA in archived samples collected in FTA cards. A statistically significant correlation was found for serotype 1 (r = 0.96), serotype 2 (r = 1), and serotype 3 (r = 0.9). The results show that FTA cards are an excellent media to collect, transport, and archive samples for MD diagnosis and to monitor MD vaccines. In addition, FTA cards are widely available, inexpensive, and adequate for the shipment of samples nationally and internationally.

Molecular Detection and Serotyping of Infectious Bronchitis Virus from FTA®Filter Paper

We investigated the feasibility of using Flinders Technology Associates (FTA) filter cards for the storage of allantoic fluid containing an infectious bronchitis virus (IBV), such as Arkansas-DPI, Connecticut, and Massachusetts, and for their identification by reverse transcriptase (RT)-polymerase chain reaction (PCR) and characterization by restriction fragment length polymorphism (RFLP) or nucleotide sequencing. FTA paper is a cotton-based cellulose membrane containing lyophilized chemicals that lyses many types of bacteria and viruses. IBV was inactivated upon contact with the FTA, as shown by the inability of the virus to be propagated in embryonating chicken eggs. RT-PCR of the S1 gene showed that viral RNA in allantoic fluid remained stable after storage on FTA filter cards and that the stability was time and temperature sensitive for the large (1700 base pair [bp]) but not the small (383 bp) PCR products. Analysis of the amplified products showed that molecular characterization is feasible in allantoic fluid stored on FTA under nonfavorable environmental conditions (41 C) for at least 15 days. The use of FTA cards for the collection, transport, and storage of IBV-containing samples is safe, inexpensive, and adequate for molecular diagnosis. We propose that specimens coming from overseas on FTA cards would be first analyzed by RT-PCR with primers yielding a 1700-bp product followed by RFLP of the positive cases. Negative cases would be analyzed with primers yielding a 383-bp product (to exdude detrimental effect of the storage conditions) followed by nucleotide sequencing of the positive cases.