Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability

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Abstract

Background

The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations.

Methods

DNA was extracted from two RDT devices (Paracheck-Pf® and SD Bioline Malaria Pf/Pan®), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman® 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance.

Results

The P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples.

Conclusions

The results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.

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Most cited references 11

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Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

Ric Price, Anne-Catrin Uhlemann, Alan Brockman … (2004)

The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days' artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.

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Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of plasmodium by amplifying RNA and DNA of the 18S rRNA genes.

Bernard Ogutu, LaDonna Tolbert, Luke Kortepeter … (2011)

A highly sensitive genus-specific quantitative reverse transcriptase real-time PCR (qRT-PCR) assay for detection of Plasmodium has been developed. The assay amplifies total nucleic acids (RNA and DNA) of the 18S rRNA genes with a limit of detection of 0.002 parasite/μl using cultured synchronized ring stage 3D7 parasites. Parasite densities as low as 0.000362 parasite/μl were detected when analyzing clinical samples. Analysis of clinical samples showed that detection of 18S rRNA genes from total nucleic acids increased the analytical sensitivity of the assay by more than 1 log unit compared to DNA only. When clinical samples with no parasites present by microscopy were analyzed by qRT-PCR, 90% (117 of 130) were positive for the presence of Plasmodium nucleic acids. Quantification of clinical samples by qRT-PCR using total nucleic acid versus DNA was compared to microscopy. There was a significantly greater correlation of parasite density to microscopy when DNA alone was used than with total nucleic acid. We conclude that analysis of total nucleic acids by qRT-PCR is a suitable assay for detection of low parasite levels in patients with early-stage malaria and/or submicroscopic infections and could greatly benefit malaria diagnosis, intervention trials, and malaria control and elimination efforts.

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Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers

Nicolas Steenkeste, Sandra Incardona, Sophy Chy … (2009)

Background Several strategies are currently deployed in many countries in the tropics to strengthen malaria control toward malaria elimination. To measure the impact of any intervention, there is a need to detect malaria properly. Mostly, decisions still rely on microscopy diagnosis. But sensitive diagnosis tools enabling to deal with a large number of samples are needed. The molecular detection approach offers a much higher sensitivity, and the flexibility to be automated and upgraded. Methods Two new molecular methods were developed: dot18S, a Plasmodium-specific nested PCR based on the 18S rRNA gene followed by dot-blot detection of species by using species-specific probes and CYTB, a Plasmodium-specific nested PCR based on cytochrome b gene followed by species detection using SNP analysis. The results were compared to those obtained with microscopic examination and the "standard" 18S rRNA gene based nested PCR using species specific primers. 337 samples were diagnosed. Results Compared to the microscopy the three molecular methods were more sensitive, greatly increasing the estimated prevalence of Plasmodium infection, including P. malariae and P. ovale. A high rate of mixed infections was uncovered with about one third of the villagers infected with more than one malaria parasite species. Dot18S and CYTB sensitivity outranged the "standard" nested PCR method, CYTB being the most sensitive. As a consequence, compared to the "standard" nested PCR method for the detection of Plasmodium spp., the sensitivity of dot18S and CYTB was respectively 95.3% and 97.3%. Consistent detection of Plasmodium spp. by the three molecular methods was obtained for 83% of tested isolates. Contradictory results were mostly related to detection of Plasmodium malariae and Plasmodium ovale in mixed infections, due to an "all-or-none" detection effect at low-level parasitaemia. Conclusion A large reservoir of asymptomatic infections was uncovered using the molecular methods. Dot18S and CYTB, the new methods reported herein are highly sensitive, allow parasite DNA extraction as well as genus- and species-specific diagnosis of several hundreds of samples, and are amenable to high-throughput scaling up for larger sample sizes. Such methods provide novel information on malaria prevalence and epidemiology and are suited for active malaria detection. The usefulness of such sensitive malaria diagnosis tools, especially in low endemic areas where eradication plans are now on-going, is discussed in this paper.

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Author and article information

Contributors

Ulrika Morris

Berit Aydin-Schmidt

Delér Shakely

Andreas Mårtensson

Louise Jörnhagen

Abdullah S Ali

Mwinyi I Msellem

Max Petzold

José P Gil

Pedro Ferreira

Anders Björkman

Journal

Journal ID (nlm-ta): Malar J

Journal ID (iso-abbrev): Malar. J

Title: Malaria Journal

Publisher: BioMed Central

ISSN (Electronic): 1475-2875

Publication date Collection: 2013

Publication date (Electronic): 19 March 2013

Volume: 12

Page: 106

Affiliations

[1 ]Malaria Research, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden

[2 ]Department of Medicine, Kungälv Hospital, Kungälv, Sweden

[3 ]Department of Public Health Sciences, Division of Global Health (IHCAR), Karolinska Institutet, Stockholm, Sweden

[4 ]Zanzibar Malaria Control Programme (ZMCP), Ministry of Health, Zanzibar, Tanzania

[5 ]Centre for Applied Biostatistics, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

[6 ]Drug Resistance and Pharmacogenetics Group, Institute of Biotechnology and Bioengineering, Centre of Molecular and Structural Biomedicine, University of Algarve, Faro, Portugal

[7 ]Laboratory of Molecular Anthropology and Health, Department of Anthropology, Binghamton University, Binghamton, NY, USA

[8 ]Department of Physiology and Pharmacology, Drug Resistance Unit, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden

[9 ]Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan

Article

Publisher ID: 1475-2875-12-106

DOI: 10.1186/1475-2875-12-106

PMC ID: 3605315

PubMed ID: 23510231

SO-VID: 7f69f597-faf5-4d60-a396-bdfb3926136e

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

History

Date received : 21 December 2012

Date accepted : 10 March 2013

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Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability

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Abstract

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Malaria vaccine development

Most cited references 11

Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of plasmodium by amplifying RNA and DNA of the 18S rRNA genes.

Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers

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Cited by 27