Dataset Information

Ultrasensitive qPCR-Based Detection of Plasmodium falciparum in Pregnant Women Using Dried Blood or Whole Blood Pellet Samples Processed through Different DNA Extraction Methods.

ABSTRACT: Highly sensitive molecular techniques for the detection of low-level Plasmodium falciparum parasitemia are highly useful for various clinical and epidemiological studies. However, differences in how blood samples are preserved, the quantity of blood stored, as well as genomic DNA extraction methods used may compromise the potential usefulness of these methodologies. This study compared diagnostic sensitivity based on microscopy and malaria rapid diagnostic tests (mRDTs), with quantitative polymerase chain reaction (qPCR) P. falciparum positivity of dried blood spots (DBS) or whole blood pellets (WBP) from pregnant women using different DNA extraction protocols (Chelex-saponin or a commercial kit). Samples from 129 pregnant women were analyzed, of which 13 were P. falciparum positive by mRDT and 5 by microscopy. By using extraction kit on WBP and on DBS, qPCR positivity was 27 (20.9%) and 16 (12.4%), respectively, whereas Chelex extraction on DBS only resulted in 4 (3.1%) P. falciparum positive samples. Thus, extraction using commercial kits greatly improve the likelihood of detecting P. falciparum infections.

SUBMITTER: Saidi Q

PROVIDER: S-EPMC8922499 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Ultrasensitive qPCR-Based Detection of Plasmodium falciparum in Pregnant Women Using Dried Blood or Whole Blood Pellet Samples Processed through Different DNA Extraction Methods.

Saidi Queen Q Minja Daniel D Njau Judith J Hansson Helle H Kavishe Reginald R Alifrangis Michael M

The American journal of tropical medicine and hygiene 20211206 3

Highly sensitive molecular techniques for the detection of low-level Plasmodium falciparum parasitemia are highly useful for various clinical and epidemiological studies. However, differences in how blood samples are preserved, the quantity of blood stored, as well as genomic DNA extraction methods used may compromise the potential usefulness of these methodologies. This study compared diagnostic sensitivity based on microscopy and malaria rapid diagnostic tests (mRDTs), with quantitative polyme ...[more]

PMID: 34872057

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Project description:BackgroundGreater Mekong Subregion countries are committed to eliminating Plasmodium falciparum malaria by 2025. Current elimination interventions target infections at parasite densities that can be detected by standard microscopy or rapid diagnostic tests (RDTs). More sensitive detection methods have been developed to detect lower density "asymptomatic" infections that may represent an important transmission reservoir. These ultrasensitive polymerase chain reaction (usPCR) tests have been used to identify target populations for mass drug administration (MDA). To date, malaria usPCR tests have used either venous or capillary blood sampling, which entails complex sample collection, processing and shipping requirements. An ultrasensitive method performed on standard dried blood spots (DBS) would greatly facilitate the molecular surveillance studies needed for targeting elimination interventions.MethodsA highly sensitive method for detecting Plasmodium falciparum and P. vivax 18S ribosomal RNA from DBS was developed by empirically optimizing nucleic acid extraction conditions. The limit of detection (LoD) was determined using spiked DBS samples that were dried and stored under simulated field conditions. Further, to assess its utility for routine molecular surveillance, two cross-sectional surveys were performed in Myanmar during the wet and dry seasons.ResultsThe lower LoD of the DBS-based ultrasensitive assay was 20 parasites/mL for DBS collected on Whatman 3MM filter paper and 23 parasites/mL for Whatman 903 Protein Saver cards-equivalent to 1 parasite per 50 µL DBS. This is about 5000-fold more sensitive than standard RDTs and similar to the LoD of ≤16-22 parasites/mL reported for other ultrasensitive methods based on whole blood. In two cross-sectional surveys in Myanmar, nearly identical prevalence estimates were obtained from contemporaneous DBS samples and capillary blood samples collected during the wet and dry season.ConclusionsThe DBS-based ultrasensitive method described in this study shows equal sensitivity as previously described methods based on whole blood, both in its limit of detection and prevalence estimates in two field surveys. The reduced cost and complexity of this method will allow for the scale-up of surveillance studies to target MDA and other malaria elimination interventions, and help lead to a better understanding of the epidemiology of low-density malaria infections.

Project description:BackgroundScreening malaria-specific antibody responses on protein microarrays can help identify immune factors that mediate protection against malaria infection, disease, and transmission, as well as markers of past exposure to both malaria parasites and mosquito vectors. Most malaria protein microarray work has used serum as the sample matrix, requiring prompt laboratory processing and a continuous cold chain, thus limiting applications in remote locations. Dried blood spots (DBS) pose minimal biohazard, do not require immediate laboratory processing, and are stable at room temperature for transport, making them potentially superior alternatives to serum. The goals of this study were to assess the viability of DBS as a source for antibody profiling and to use DBS to identify serological signatures of low-density Plasmodium falciparum infections in malaria-endemic regions of Myanmar.MethodsMatched DBS and serum samples from a cross-sectional study in Ingapu Township, Myanmar were probed on protein microarrays populated with P. falciparum antigen fragments. Signal and trends in both sample matrices were compared. A case-control study was then performed using banked DBS samples from malaria-endemic regions of Myanmar, and a regularized logistic regression model was used to identify antibody signatures of ultrasensitive PCR-positive P. falciparum infections.ResultsApproximately 30% of serum IgG activity was recovered from DBS. Despite this loss of antibody activity, antigen and population trends were well-matched between the two sample matrices. Responses to 18 protein fragments were associated with the odds of asymptomatic P. falciparum infection, albeit with modest diagnostic characteristics (sensitivity 58%, specificity 85%, negative predictive value 88%, and positive predictive value 52%).ConclusionsMalaria-specific antibody responses can be reliably detected, quantified, and analysed from DBS, opening the door to serological studies in populations where serum collection, transport, and storage would otherwise be impossible. While test characteristics of antibody signatures were insufficient for individual diagnosis, serological testing may be useful for identifying exposure to asymptomatic, low-density malaria infections, particularly if sero-surveillance strategies target individuals with low previous exposure as sentinels for population exposure.

Dataset Information

Ultrasensitive qPCR-Based Detection of Plasmodium falciparum in Pregnant Women Using Dried Blood or Whole Blood Pellet Samples Processed through Different DNA Extraction Methods.

Publications

Ultrasensitive qPCR-Based Detection of Plasmodium falciparum in Pregnant Women Using Dried Blood or Whole Blood Pellet Samples Processed through Different DNA Extraction Methods.

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