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Genotyping Plasmodium falciparum gametocytes using amplicon deep sequencing.
Vareta, Jimmy; Horstman, Natalie A; Adams, Matthew; Seydel, Karl B; McCann, Robert S; Cohee, Lauren M; Laufer, Miriam K; Takala-Harrison, Shannon.
Affiliation
  • Vareta J; Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, USA.
  • Horstman NA; Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, USA.
  • Adams M; Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
  • Seydel KB; Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, USA.
  • McCann RS; Blantyre Malaria Project, Kamuzu University of Health Sciences, Blantyre, Malawi.
  • Cohee LM; College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA.
  • Laufer MK; Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, USA.
  • Takala-Harrison S; Center for Vaccine Development and Global Health, University of Maryland School of Medicine, Baltimore, MD, USA.
Malar J ; 23(1): 96, 2024 Apr 06.
Article de En | MEDLINE | ID: mdl-38582837
ABSTRACT

BACKGROUND:

Understanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing.

METHODS:

A data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach.

RESULTS:

A 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI 0.92-0.99] and 0.92 [95% CI 0.83-0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI 0.28-0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI 1.35-3.68].

CONCLUSIONS:

A region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.
Sujet(s)
Mots clés

Texte intégral: 1 Collection: 01-internacional Base de données: MEDLINE Sujet principal: Plasmodium falciparum / Paludisme à Plasmodium falciparum Limites: Female / Humans / Male Langue: En Journal: Malar J Sujet du journal: MEDICINA TROPICAL Année: 2024 Type de document: Article Pays d'affiliation: États-Unis d'Amérique

Texte intégral: 1 Collection: 01-internacional Base de données: MEDLINE Sujet principal: Plasmodium falciparum / Paludisme à Plasmodium falciparum Limites: Female / Humans / Male Langue: En Journal: Malar J Sujet du journal: MEDICINA TROPICAL Année: 2024 Type de document: Article Pays d'affiliation: États-Unis d'Amérique
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