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

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 μl compared to 20 μl or 30 μl 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.