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A Polymerase Chain Reaction/Ligase Detection Reaction–Fluorescent Microsphere Assay to Determine Plasmodium falciparum MSP-1₁₉ Haplotypes

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-1₁₉ 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-1₁₉) 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-1₁₉ allele-specific polymerase chain reaction (PCR)/ligase detection reaction–fluorescent microsphere assay (LDR-FMA) that allows simultaneous detection of the four predominant MSP-1₁₉ 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-1₁₉ haplotypes in epidemiologic studies.

Received January 2, 2007. Accepted for publication April 27, 2007.

Acknowledgments: This manuscript was published with permission of the Director of the Kenya Medical Research Institute. We thank the study participants and Kenyan field and laboratory technicians.

Financial support: This research was funded by NIH R01 AI43906 (JK), K08 AI51565 (AM), and T32 AI0702427 (AD).

^* Address correspondence to Ann M. Moormann, Center for Global Health and Diseases, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Wolstein Research Building 4-130, Cleveland, OH 44106-7286. E-mail: moorms{at}case.edu

Authors’ addresses: Arlene Dent, Christopher Yohn, Pete Zimmerman, James W. Kazura, Ann M. Moormann, Center for Global Health & Diseases, Case Western Reserve University, 2103 Cornell Rd 4-142, Wolstein Research Building, Cleveland, OH 44106-7286, Telephone: 216-368-6005, Fax: 216-368-4825, E-mails: arlene.dent{at}case.edu, Christopher.yohn{at}case.edu, paz{at}case.edu, jxk14{at}case.edu, and moorms{at}case.edu. John Vulule, Center for Vector Biology & Control Research, Kenya Medical Research Institute, PO Box 1578, Kisumu, Kenya, Telephone: 254-57-202-2989, E-mail: JVulule{at}kisian.mimcom.net.