Laser Cutting Eliminates Nucleic Acid Cross-Contamination in Dried-Blood-Spot Processing

Murphy, Sean C.; Daza, Glenda; Chang, Ming; Coombs, Robert W.

doi:10.1128/jcm.02549-12

Cited by 38 publications

(51 citation statements)

References 13 publications

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“…This study was the first to demonstrate accurate parasite detection and quantification by this qRT-PCR method. Because the 18S rRNA is biologically enriched in malaria-infected erythrocytes relative to the parent 18S rDNA by a factor of 3500–5000 [16] , [25] , the qRT-PCR format offers robust sensitivity relative to qPCR for samples of the same volume.…”

Section: Discussionmentioning

confidence: 99%

Safety and Comparability of Controlled Human Plasmodium falciparum Infection by Mosquito Bite in Malaria-Naïve Subjects at a New Facility for Sporozoite Challenge

et al. 2014

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BackgroundControlled human malaria infection (CHMI) studies which recapitulate mosquito-borne infection are a critical tool to identify protective vaccine and drug candidates for advancement to field trials. In partnership with the Walter Reed Army Institute of Research, the CHMI model was established at the Seattle Biomedical Research Institute's Malaria Clinical Trials Center (MCTC). Activities and reagents at both centers were aligned to ensure comparability and continued safety of the model. To demonstrate successful implementation, CHMI was performed in six healthy malaria-naïve volunteers.MethodsAll volunteers received NF54 strain Plasmodium falciparum by the bite of five infected Anopheles stephensi mosquitoes under controlled conditions and were monitored for signs and symptoms of malaria and for parasitemia by peripheral blood smear. Subjects were treated upon diagnosis with chloroquine by directly observed therapy. Immunological (T cell and antibody) and molecular diagnostic (real-time quantitative reverse transcriptase polymerase chain reaction [qRT-PCR]) assessments were also performed.ResultsAll six volunteers developed patent parasitemia and clinical malaria. No serious adverse events occurred during the study period or for six months post-infection. The mean prepatent period was 11.2 days (range 9–14 days), and geometric mean parasitemia upon diagnosis was 10.8 parasites/µL (range 2–69) by microscopy. qRT-PCR detected parasites an average of 3.7 days (range 2–4 days) earlier than blood smears. All volunteers developed antibodies to the blood-stage antigen merozoite surface protein 1 (MSP-1), which persisted up to six months. Humoral and cellular responses to pre-erythrocytic antigens circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) were limited.ConclusionThe CHMI model was safe, well tolerated and characterized by consistent prepatent periods, pre-symptomatic diagnosis in 3/6 subjects and adverse event profiles as reported at established centers. The MCTC can now evaluate candidates in the increasingly diverse vaccine and drug pipeline using the CHMI model.Trial RegistrationClinicalTrials.gov NCT01058226

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Section: Discussionmentioning

confidence: 99%

Safety and Comparability of Controlled Human Plasmodium falciparum Infection by Mosquito Bite in Malaria-Naïve Subjects at a New Facility for Sporozoite Challenge

et al. 2014

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“…In contrast, in the University of Washington (UW) qRT-PCR method [ 14 ], ~3,500 target 18S rRNA copies are present per ring-stage parasite. At the 20 p/mL published LLD, the quanta of rRNA for a single parasite in 0.05 mL of whole blood leads to qRT-PCR detection at a C T of ~33.5 cycles.…”

Section: Introductionmentioning

confidence: 99%

Increased sample volume and use of quantitative reverse-transcription PCR can improve prediction of liver-to-blood inoculum size in controlled human malaria infection studies

Hodgson

Douglas

Edwards

et al. 2015

Malar J

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BackgroundControlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants. The lower limits of detection and quantification vary amongst the assays used throughout the world, which may affect the ability of mathematical models to accurately estimate the liver-to-blood inoculum (LBI) values that are used to judge the efficacy of pre-erythrocytic vaccine and drug candidates.MethodsSamples were collected around the time of onset of pre-patent parasitaemia from subjects who enrolled in two different CHMI clinical trials. Blood samples were tested for Plasmodium falciparum 18S rRNA and/or rDNA targets by different NAT methods and results were compared. Methods included an ultrasensitive, large volume modification of an established quantitative reverse transcription PCR (qRT-PCR) assay that achieves detection of as little as one parasite/mL of whole blood.ResultsLarge volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology. Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI. In these trials, the proportion of participants for whom LBI could be accurately quantified using parasite density value greater than or equal to the lower limit of quantification was increased. A greater improvement would be expected in trials in which numerous subjects receive a lower LBI or low dose challenge.ConclusionsStandard qPCR and qRT-PCR methods with analytical sensitivities of ~20 parasites/mL probably suffice for most CHMI purposes, but the newly developed large volume qRT-PCR may be able to answer specific questions when more analytical sensitivity is required.

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“…One DBS was excised using a laser cutter [9] and soaked in 2.5 mL lysis buffer (bioMerieux) with agitation for two hours. The lysate then was quantified using the Abbott RealTime HIV-1 assay and the result was corrected for a dilution factor of 50 to give an estimated HIV-1 RNA copies/mL of blood [10].…”

Section: Methodsmentioning

confidence: 99%

Detection of HIV RNA in dried blood spots and oral fluids

Stekler

Ure²,

Dragavon³

et al. 2017

Aids

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We evaluated detection of HIV-1 RNA from dried blood spots (DBS) and oral fluid specimens. Between February 2010 and August 2014, HIV-1 was newly diagnosed in eight (2.6%) subjects who had median blood HIV-1 RNA of 61,500 copies/mL (IQR 7500-146,000). RNA was detected in 7 (87.5%) DBS and 3 (37.5%) oral fluid swabs but was not detected in either specimen from one participant. DBS may be a reasonable specimen collection method to detect acute infection.

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Laser Cutting Eliminates Nucleic Acid Cross-Contamination in Dried-Blood-Spot Processing

Cited by 38 publications

References 13 publications

Safety and Comparability of Controlled Human Plasmodium falciparum Infection by Mosquito Bite in Malaria-Naïve Subjects at a New Facility for Sporozoite Challenge

Safety and Comparability of Controlled Human Plasmodium falciparum Infection by Mosquito Bite in Malaria-Naïve Subjects at a New Facility for Sporozoite Challenge

Increased sample volume and use of quantitative reverse-transcription PCR can improve prediction of liver-to-blood inoculum size in controlled human malaria infection studies

Detection of HIV RNA in dried blood spots and oral fluids

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