Distinction of Plasmodium falciparum recrudescence and re-infection by MSP2 genotyping: a caution about unstandardized classification criteria.

Abstract

BACKGROUND:

Plasmodium falciparum genotyping with molecular polymorphic markers is widely employed to distinguish recrudescence from re-infection in antimalarial drug efficacy monitoring programmes. However, limitations occur on agarose gel DNA measurements used to resolve the polymorphisms. Without empirical data, the current distinction of pre- and post-treatment bands, as persistent or new infection, is subjective and often varying by author. This study measures empirical tolerance limits for classifying different-sized bands as same or different alleles during MSP2 genotyping.

METHODS:

P. falciparum field samples from 161 volunteers were genotyped by nested PCR using polymorphic MSP2 family-specific primers. Data were analysed to determine variability of band size measurements between identical MSP2 alleles randomized into different agarose lanes.

RESULTS:

The mean (95% CI) paired difference in band size between identical alleles was 9.8 bp (1.48 - 18.16 bp, p = 0.022) for 3D7/IC and 2.54 (-3.04 - 8.05 bp, p = 0.362) for FC27. Based on these findings, pre- and post-treatment samples with 3D7/IC alleles showing less than 18 bp difference corresponded to recrudescence, with 95% confidence, while greater difference indicated new infection. FC27 allele differences were much narrower. For both 3D7/IC and FC27 amplicon, allele detection sensitivity was significantly higher with 13 mul compared to 20 mul or 30 mul lane loading volumes.

CONCLUSION:

During MSP genotyping, it is useful to standardize classifications against measurement of background variability on identical alleles, in order to obtain reliable findings. It is critical to use a fixed optimal lane loading volume for constant allele patency, to avoid the disappearance or false appearance of new infection.

• Authors: Petrica Rouse, Mtawa Ap Mkulama, Philip E Thuma, Sungano Mharakurwa
• Journal: Malaria journal (Malar J) Vol. 7 Pg. 185 (Sep 23 2008) ISSN: 1475-2875 [Electronic] England
• PMID: 18811949 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antigens, Protozoan
• Protozoan Proteins
• merozoite surface protein 2, Plasmodium

Topics

• Adolescent
• Adult
• Animals
• Antigens, Protozoan (genetics)
• Child
• Child, Preschool
• DNA Fingerprinting (standards)
• Electrophoresis, Agar Gel (standards)
• Female
• Genotype
• Humans
• Malaria, Falciparum (parasitology)
• Male
• Middle Aged
• Plasmodium falciparum (classification, genetics, isolation & purification)
• Polymerase Chain Reaction (methods)
• Polymorphism, Genetic
• Protozoan Proteins (genetics)
• Recurrence
• Reproducibility of Results