Molecular methods in malaria control in the era of pre-elimination

Abstract

Increased funding combined with effective malaria control methods for prevention, diagnosis, and treatment, has resulted in a 30% reduction of the global malaria burden over the last decade. As malaria prevalence declines in areas of successful malaria control, the proportion of subpatent infections that fall below the detection level of malaria rapid diagnostic tests (RDTs) and microscopy increases. In these areas new tools and strategies are required for detecting and targeting residual parasite populations. Furthermore, there is an emerging resistance to the artemisinin-based combination therapies (ACTs), which is the recommended first-line treatment for uncomplicated Plasmodium falciparum malaria. This resistance is a serious threat to the recent achievements in the reduction of the global malaria burden.

The aim of this thesis was to gain insight into the application of modern molecular methods for enhanced malaria infection detection and surveillance of antimalarial drug resistance in a pre-elimination setting such as Zanzibar.

Study I assessed whether seven years of wide scale use of artesunate-amodiaquine (ASAQ) as first-line treatment selected for P. falciparum single nucleotide polymorphisms (SNPs) associated with resistance to the ACT partner drug amodiaquine. No selection of SNPs associated with amodiaquine resistance was observed, indicating sustained efficacy of ASAQ as first-line treatment in Zanzibar.

In Study II different methods of DNA extraction from used RDTs were evaluated and it was assessed whether RDTs could preserve Plasmodium DNA for the purpose of molecular epidemiological investigations. The Chelex-100 method proved the most sensitive method of DNA extraction in both RDT and filter paper samples. RDTs collected in Zanzibar provided parasite DNA of equal quality as filter papers, suggesting that RDTs are a valuable alternative for DNA storage under field conditions.

In Study III a highly sensitive SYBR Green qPCR-RFLP assay was developed for Plasmodium detection and species determination in samples collected on filter paper. This method was applied in Study IV for characterising asymptomatic Plasmodium infections. A declining, albeit persistent, reservoir of parasites present at low-densities was found in asymptomatic individuals, highlighting the need for sensitive molecular methods in malaria pre-elimination settings. The study revealed important characteristics of the remaining parasite populations, including intriguing trends in SNPs associated with antimalarial drug resistance that require further investigation in order to be fully understood.

Study V reports the hitherto largest implementation of a new molecular diagnostic tool based on loop-mediated isothermal amplification (LAMP), for scaled up, centralised mass-screening of asymptomatic malaria in Zanzibar. LAMP detected 3.4 times more Plasmodium positive samples than RDT, and was found to be a simple and sensitive molecular tool with potential for use in active malaria surveillance. Contamination is, however, a concern. A higher throughput, affordable closed system would be ideal to avoid DNA contamination when processing larger numbers of samples.

In summary, molecular methods are required for enhanced malaria infection detection and surveillance of antimalarial drug resistance in malaria pre-elimination settings such as Zanzibar. The application of molecular methods may be of particular interest for malaria control/elimination programs, for monitoring progress towards malaria elimination and for optimal orientation of program activities.