Targeted and whole-genome sequencing reveal a north-south divide in P. falciparum drug resistance markers and genetic structure in Mozambique

Mozambique is one of the four African countries which account for over half of all malaria deaths worldwide, yet little is known about the parasite genetic structure in that country. We performed P. falciparum amplicon and whole genome sequencing on 2251 malaria-infected blood samples collected in 2015 and 2018 in seven provinces of Mozambique to genotype antimalarial resistance markers and interrogate parasite population structure using genome-wide microhaplotyes. Here we show that the only resistance-associated markers observed at frequencies above 5% were pfmdr1-184F (59%), pfdhfr-51I/59 R/108 N (99%) and pfdhps-437G/540E (89%). The frequency of pfdhfr/pfdhps quintuple mutants associated with sulfadoxine-pyrimethamine resistance increased from 80% in 2015 to 89% in 2018 (p < 0.001), with a lower expected heterozygosity and higher relatedness of microhaplotypes surrounding pfdhps mutants than wild-type parasites suggestive of recent selection. pfdhfr/pfdhps quintuple mutants also increased from 72% in the north to 95% in the south (2018; p < 0.001). This resistance gradient was accompanied by a concentration of mutations at pfdhps-436 (17%) in the north, a south-to-north increase in the genetic complexity of P. falciparum infections (p = 0.001) and a microhaplotype signature of regional differentiation. The parasite population structure identified here offers insights to guide antimalarial interventions and epidemiological surveys.

Enhanced T cell activation in Plasmodium falciparum malaria-infected human immunodeficiency virus-1 patients from Mozambique

Human immunodeficiency virus (HIV)-1 infection has an important impact on malaria. Plasmodium falciparum and HIV-1 co-infected patients (Pf/HIV) present with a high degree of anaemia, enhanced parasitaemia and decreased CD4+ T cell counts, which increase the risk of developing severe malaria. In addition, infection with either Pf or HIV-1 alone causes extensive immune activation. Our hypothesis was that lymphocyte activation is potentiated in Pf/HIV co-infected patients, consequently worsening their immunosuppressed state. To test this hypothesis, 22 Pf/HIV patients, 34 malaria patients, 29 HIV/AIDS patients and 10 healthy controls without malaria or HIV/acquired immune deficiency syndrome (AIDS) from Maputo/Mozambique were recruited for this study. As expected, anaemia was most prevalent in the Pf/HIV group. A significant variation in parasite density was observed in the Pf/HIV co-infected group (110-75,000 parasites/µL), although the median values were similar to those of the malaria only patients. The CD4+ T cell counts were significantly lower in the Pf/HIV group than in the HIV/AIDS only or malaria only patients. Lymphocyte activation was evaluated by the percentage of activation-associated molecules [CD38 expression on CD8+ and human leukocyte antigen-DR expression on CD3+ T cells]. The highest CD38 expression was detected in the Pf/HIV co-infected patients (median = 78.2%). The malaria only (median = 50%) and HIV/AIDS only (median = 52%) patients also exhibited elevated levels of these molecules, although the values were lower than those of the Pf/HIV co-infected cases. Our findings suggest that enhanced T-cell activation in co-infected patients can worsen the immune response to both diseases.

Antimalarial drug resistance in Southern Mozambique: treatment efficacy molecular characterization of Plasmodium falciparum resistance to sulfadoxina-pyrimathamine

Resistance of Plasmodium falciparum to antimalarial drugs is widespread in many countries in Africa; including Mozambique; and it makes the control of malaria difficult. Sulfadoxina-pyrimethamine (SP) is the most common alternative; used as first-or second-line drug in malaria endemic areas where the parasites are tesistant to chloroquine (CQ). However; there is a concern that the extensive use of SP may lead to rapid emergency of P. falciparum resistant strains. Using molecular markers that have been correlated to SP resistance; we carried out the studies presented in this thesis; where the mai objective was to assess the levels of resistance and to study the mocecular markers that underline the dynamics of resistance to sulfadoxine-pyrimethamine. The studies were conducted in Manhica district; an area of intense and perennial malarial transmission in southern Mozambique. Chapter 1 gives ageneral introduction; the rationale and the objective of the study. In chapter 2 the background informationof the study area; the study population; study design and the laboratory procedures used are presented; with more emphasis on the methods used to identify the genotypes in the P. falciparum dhfr/dhps genes. The findings of the field studies are reported in chapters 3 to 6 as summarized below. Chapter 3 reports results of the study on the efficacy of chloroquine (CQ); sufadoxine-pyrimethamine (SP) and amodiaquine(AQ) among children with incomplicated malaria. Chapter 4 describe results of the study investigating the correlation between the presence of mutations in the dhfr and dhps genes of P falciparum and the in vivi outcome following SP traetment of children under five years of age in Manhica district; southern Mozambique. Chapter 5 present the results of a study investigating the frequency of mutation halotypes in the dhfr and dhps genes of P. falciparum; at the community level. Chapter 6 presents results of a study exploring mothers' treatment-seeking behavioral regarding fever and childhood malaria and assessing the level of usage of antimalarial drugs by the community. Chapter 7 discuss the main findings of all chapters; summarizes the conclusions of all studies and discusses areas for future research