A polymerase chain reaction/ligase detection reaction fluorescent microsphere assay to determine Plasmodium falciparum MSP-119 haplotypes

Abstract

The merozoite surface protein-1 (MSP-1) is a blood stage antigen currently being tested as a vaccine against Plasmodium falciparum malaria. Determining the MSP-1(19) haplotype(s) present during infection is essential for assessments of MSP-1 vaccine efficacy and studies of protective immunity in human populations. The C-terminal fragment (MSP-1(19)) has four predominant haplotypes based on point mutations resulting in non-synonymous amino acid changes: E-TSR (PNG-MAD20 type), E-KNG (Uganda-PA type), Q-KNG (Wellcome type), and Q-TSR (Indo type). Current techniques using direct DNA sequencing are laborious and expensive. We present an MSP-1(19) allele-specific polymerase chain reaction (PCR)/ligase detection reaction-fluorescent microsphere assay (LDR-FMA) that allows simultaneous detection of the four predominant MSP-1(19) haplotypes with a sensitivity and specificity comparable with other molecular methods and a semi-quantitative determination of haplotype contribution in mixed infections. Application of this method is an inexpensive, accurate, and high-throughput alternative to distinguish the predominant MSP-1(19) haplotypes in epidemiologic studies.

Figure 1

Allele-specific microsphere MFI averaged from 16 separate MSP-1₁₉ PCR/LDR-MFA experiments. Controls include ETSR only, QTSR only, and water. ETSR only represents MSP-1₁₉ PCR/LDR-MFA performed with DNA from 3D7 parasites. QKNG only represents MSP-1₁₉ PCR/LDR-MFA performed with DNA from K1 parasites. A 1:1 combination of 3D7 and K1 parasite DNA was serially diluted from 1:10 to 1:10,000. The positive threshold for Q- and E-associated microspheres was set at 800 MFI (dashed horizontal line) and 3,000 MFI for KNG and TSR (solid horizontal line). The positive threshold for Pf specific associated microsphere was set at 3,000 MFI. Determination of positive alleles was made using 35-cycle PCR products.

Figure 2

Relative allele contribution. Known amounts of cultured parasite DNA from strains 3D7 (ETSR) and K1 (QKNG) were combined at various concentrations (i.e., 3xQKNG:1xETSR, 2xQKNG:1xETSR, 1xQKNG:1xETSR) and measured at 27 cycles of MSP-1₁₉–specific PCR/LDR-FMA. A, MFIs of KNG and TSR mi-crospheres increase or decrease in direct relation to the relative amount of each haplotype. B, MFIs of Q and E microspheres do not increase or decrease in direct relation to the relative amount of each haplotype in the experimental sample. C, However, if the Q MFI is doubled, the expected result occurs and the relative Q:E contributions can be more directly discerned.

Figure 3

Relative MFI of comparative Luminex microspheres for each allele tested using Kenyan samples. An arbitrary dashed line was drawn intersecting the Q and E allele MFI axes showing the relative differences in MFI for each microsphere: a “Q” MFI of 2,000 was equivalent to an “E” MFI of 5,000. Using this comparative scale, the relative contribution of each allele to the Pf infection was determined. In contrast, the KNG- and TSR-associated microspheres had a similar MFI range, and therefore direct comparisons of relative intensity could be made. A positive correlation between allele MFI and Pf MFI can be observed.