Dataset Information

Bead-based assays to simultaneously detect multiple human inherited blood disorders associated with malaria.

ABSTRACT:

Background

Glucose-6-phosphate dehydrogenase deficiency (G6PDd), haemoglobin C (HbC) and S (HbS) are inherited blood disorders (IBD) common in populations in malaria endemic areas. All are associated to some degree with protection against clinical malaria whilst additionally G6PDd is associated with haemolysis following treatment with 8-aminoquinolines. Measuring the prevalence of these inherited blood disorders in affected populations can improve understanding of disease epidemiology. Current methodologies in epidemiological studies commonly rely on individual target amplification and visualization; here a method is presented to simultaneously detect the polymorphisms and that can be expanded to include other single nucleotide polymorphisms (SNPs) of interest.

Methods

Human DNA from whole blood samples was amplified in a novel, multiplex PCR reaction and extended with SNP-specific probes in an allele specific primer extension (ASPE) to simultaneously detect four epidemiologically important human markers including G6PD SNPs (G202A and A376G) and common haemoglobin mutations (HbS and HbC). The products were hybridized to magnetic beads and the median fluorescence intensity (MFI) was read on MAGPIX^® (Luminex corp.). Genotyping data was compared to phenotypical data generated by flow cytometry and to established genotyping methods.

Results

Seventy-five samples from Burkina Faso (n?=?75/78, 96.2%) and 58 samples from The Gambia (n?=?58/61, 95.1%) had a G6PD and a HBB genotype successfully assigned by the bead-based assay. Flow cytometry data available for n?=?61 samples further supported the concordance between % G6PD normal/deficient cells and genotype.

Conclusions

The bead based assay compares well to alternative measures of genotyping and phenotyping for G6PD. The screening is high throughput, adaptable to inclusion of multiple targets of interest and easily standardized.

SUBMITTER: Grignard L

PROVIDER: S-EPMC6341711 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Bead-based assays to simultaneously detect multiple human inherited blood disorders associated with malaria.

Grignard Lynn L Mair Catherine C Curry Jonathan J Mahey Laleta L Bastiaens Guide J H GJH Tiono Alfred B AB Okebe Joseph J Coulibaly Sam A SA Gonçalves Bronner P BP Affara Muna M Ouédraogo Alphonse A Bougouma Edith C EC Sanou Guillaume S GS Nébié Issa I Lanke Kjerstin H W KHW Sirima Sodiomon B SB d'Alessandro Umberto U Clark Taane G TG Campino Susana S Bousema Teun T Drakeley Chris C

Malaria journal 20190121 1

<h4>Background</h4>Glucose-6-phosphate dehydrogenase deficiency (G6PDd), haemoglobin C (HbC) and S (HbS) are inherited blood disorders (IBD) common in populations in malaria endemic areas. All are associated to some degree with protection against clinical malaria whilst additionally G6PDd is associated with haemolysis following treatment with 8-aminoquinolines. Measuring the prevalence of these inherited blood disorders in affected populations can improve understanding of disease epidemiology. C ...[more]

PMID: 30665411

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Project description:BackgroundMalaria remains an important public health problem in Latin America, and the development of insecticide resistance in malaria vectors poses a major threat to malaria elimination efforts. Monitoring of insecticide susceptibility and the determination of the mechanisms involved in insecticide resistance are needed to effectively guide the deployment of appropriate vector control measures. Here, molecular assays have been developed to screen for mutations associated with insecticide resistance on the voltage-gated sodium channel (VGSC) and acetylcholinesterase-1 (Ace-1) genes in four malaria vectors from Latin America.MethodsDegenerate primers were designed to amplify a partial fragment on the VGSC and Ace-1 genes. Wild-caught individuals for Anopheles albimanus (also historical samples and individuals from a laboratory strain), Anopheles darlingi, Anopheles vestitipennis and Anopheles pseudopunctipennis were used to optimize the PCR assays. All samples were sequenced to validate the PCR results and DNA alignments were constructed for each gene using the unique haplotypes observed.ResultsPrimers designed successfully amplified the VGSC gene in An. albimanus, An. darlingi, An. vestitipennis and An. pseudopunctipennis, and the Ace-1 gene in both An. albimanus and An. darlingi. DNA sequencing revealed that compared with Anopheles gambiae, there were a total of 29, 28, 21 and 24 single nucleotide polymorphisms (SNPs) on the VGSC gene for An. albimanus (308 bp), An. darlingi (311 bp), An. pseudopunctipennis (263 bp) and An. vestitipennis (254 bp), respectively. On the 459 bp fragment of the Ace-1 gene, a total of 70 SNPs were detected in An. darlingi and 59 SNPs were detected in An. albimanus compared with An. gambiae. The SNPs detected on the VGSC gene were all synonymous. On the Ace-1 gene, non-synonymous substitutions were identified on three different codons. All species showed the homozygous wild-type kdr allele (coding for leucine) at codon 995 (formerly reported as codon 1014) on the VGSC gene, but one sample was heterozygous at codon 280 (formerly reported as codon 119) on the Ace-1 gene, coding for both the resistant (serine) and susceptible (glycine) amino acids.ConclusionsNew molecular assays to amplify and screen the regions of the VGSC and Ace-1 genes associated with insecticide resistance are reported for An. albimanus, An. darlingi, An. vestitipennis, and An. pseudopunctipennis. The development of these PCR assays presents an important advance in the analysis of target-site resistance in malaria vectors in the Americas, and will further facilitate the characterization of insecticide resistance mechanisms in these species.

Dataset Information

Bead-based assays to simultaneously detect multiple human inherited blood disorders associated with malaria.

Background

Methods

Results

Conclusions

Publications

Bead-based assays to simultaneously detect multiple human inherited blood disorders associated with malaria.

Similar Datasets

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