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Malar J. 2019 Oct 2;18(1):336. doi: 10.1186/s12936-019-2972-y.

DNA recovery from archived RDTs for genetic characterization of Plasmodium falciparum in a routine setting in Lambaréné, Gabon.

Author information

1
Institute of Tropical Medicine, University of Tübingen, Wilhelmstraße 27, 72074, Tübingen, Germany. drthe108@gmail.com.
2
German Center for Infection Research, University of Tübingen, Wilhelmstraße 27, 72074, Tübingen, Germany. drthe108@gmail.com.
3
Centre de Recherches Médicales de Lambaréné and African Partner Institution, German-Center for Infection Research, Lambaréné, Gabon. drthe108@gmail.com.
4
Vietnamese-German Center for Medical Research, Hanoi, Vietnam. drthe108@gmail.com.
5
Centre de Recherches Médicales de Lambaréné and African Partner Institution, German-Center for Infection Research, Lambaréné, Gabon.
6
Institute of Tropical Medicine, University of Tübingen, Wilhelmstraße 27, 72074, Tübingen, Germany.
7
German Center for Infection Research, University of Tübingen, Wilhelmstraße 27, 72074, Tübingen, Germany.
8
Department of Tropical Medicine, Bernhard Nocht Institute for Tropical Medicine & I. Department of Medicine University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
9
Division of Infectious Diseases and Tropical Medicine, Department of Medicine I, Medical University Vienna, Vienna, Austria.
10
Vietnamese-German Center for Medical Research, Hanoi, Vietnam.
11
Fondation Congolaise pour la Recherche Médicale, Brazzaville, Congo.
12
Institute of Tropical Medicine, University of Tübingen, Wilhelmstraße 27, 72074, Tübingen, Germany. benjamin.mordmueller@uni-tuebingen.de.
13
German Center for Infection Research, University of Tübingen, Wilhelmstraße 27, 72074, Tübingen, Germany. benjamin.mordmueller@uni-tuebingen.de.
14
Centre de Recherches Médicales de Lambaréné and African Partner Institution, German-Center for Infection Research, Lambaréné, Gabon. benjamin.mordmueller@uni-tuebingen.de.

Abstract

BACKGROUND:

Rapid diagnostic tests (RDTs) have been described as a source of genetic material to analyse malaria parasites in proof-of-concept studies. The increasing use of RDTs (e.g., in focal or mass screening and treatment campaigns) makes this approach particularly attractive for large-scale investigations of parasite populations. In this study, the complexity of Plasmodium falciparum infections, parasite load and chloroquine resistance transporter gene mutations were investigated in DNA samples extracted from positive RDTs, obtained in a routine setting and archived at ambient temperature.

METHODS:

A total of 669 archived RDTs collected from malaria cases in urban, semi-urban and rural areas of central Gabon were used for P. falciparum DNA extraction. Performance of RDTs as a source of DNA for PCR was determined using: (i) amplification of a single copy merozoite surface protein 1 (msp1) gene followed by highly sensitive and automated capillary electrophoresis; (ii) genotyping of the pfcrt gene locus 72-76 using haplotype-specific-probe-based real-time PCR to characterize chloroquine resistance; and, (iii) real-time PCR targeting 18S genes to detect and quantify Plasmodium parasites.

RESULTS:

Out of the 669 archived RDTs, amplification of P. falciparum nucleic materials had a success rate of 97% for 18S real-time PCR, and 88% for the msp1 gene. The multiplicity of infections (MOI) of the whole population was 2.6 (95% CI 2.5-2.8). The highest number of alleles detected in one infection was 11. The MOI decreased with increasing age (β = - 0.0046, p = 0.02) and residence in Lambaréné was associated with smaller MOIs (p < 0.001). The overall prevalence of mutations associated with chloroquine resistance was 78.5% and was not associated with age. In Lambaréné, prevalence of chloroquine resistance was lower compared to rural Moyen-Ogooué (β = - 0.809, p-value = 0.011).

CONCLUSION:

RDT is a reliable source of DNA for P. falciparum detection and genotyping assays. Furthermore, the increasing use of RDTs allows them to be an alternative source of DNA for large-scale genetic epidemiological studies. Parasite populations in the study area are highly diverse and prevalence of chloroquine-resistant P. falciparum remains high, especially in rural areas.

KEYWORDS:

Capillary electrophoresis; Gabon; Malaria; Plasmodium falciparum; RDT; msp1; pfcrt

PMID:
31578142
DOI:
10.1186/s12936-019-2972-y
Free PMC Article

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