Luz García

: Artemisinin-combined treatments are the recommended first-line treatment of Plasmodium falciparum malaria, but they are being threatened by emerging artemisinin resistance. Mutations in pfk13 are the principal molecular marker for artemisinin resistance. This study characterizes the presence of mutations in pfk13 in P. falciparum in Western Equatoria State, South Sudan. We analyzed 468 samples from patients with symptomatic malaria and found 15 mutations (8 nonsynonymous and 7 synonymous). Each mutation appeared only once, and none were validated or candidate markers of artemisinin resistance. However, some mutations were in the same or following position of validated and candidate resistance markers, suggesting instability of the gene that could lead to resistance. The R561L nonsynonymous mutation was found in the same position as the R561H validated mutation. Moreover, the A578S mutation, which is widespread in Africa, was also reported in this study. We found a high diversity of other pfk13 mutations in low frequency. Therefore, routine molecular surveillance of resistance markers is highly recommended to promptly detect the emergence of resistance-related mutations and to limit their spread.

Background: Malaria is one of the deadliest disease in the world, particularly in Africa. The resistance to antimalarial drugs is one of the most important problems to global malaria control. The study assess the evolution of the different resistance markers over time and the possible influence of the interventions and treatment changes that have been made in Equatorial Guinea.Methods: 1223 biological samples distributed from 1999 to 2019 were included in the study. The screening of the mutations in pfdhfr, pfdhps, pfmdr1 and pfcrt genes were carried out by nested PCR and RFLPs, and the study of pfk13 genes was carried out by nested PCR, followed by sequencing to determine the presence of mutations Results: the haplotypes partially and fully resistant (pfdhfr + pfdhps) increase over time. In 2019, the fully resistant is increasing although super resistant remains lower. There is also a continued decline over time in pfmdr1 and pfcrt gene mutations. Since 2008, when ACTs were introduced in the country, the number of mutations detected in pfk13 has been increasing over time, with more mutations being observed in 2019. In this year, were detected 2 synonymous and 5 non-synonymous mutations, although are not related to resistance to ACTs. In addition, the non-synonymous A578S mutation, the most frequent on the African continent, was detected in 2013, although it has not been detected in the following years.Conclusions: The withdrawal of the use of CQ as a treatment in the country has been effective over time, as wild-type parasite populations outnumber mutant populations. The upward trend observed in SP resistance markers evidence its misuse alone or in combination with AS or AQ in some areas of the country, this allows selective pressure from SP to continue. SNPs 540E and 581G do not exceed the limit of 50% and 10% respectively, which means that SP as an IPT is still effective in the country. As for the pfk13 gene, no mutations have been detected in relation to resistance to ACTs. However, in 2019 there is a greater accumulation of non-synonymous mutations compared to years prior to 2008.

The emergence of drug resistance in Plasmodium falciparum has been a major contributor to the global burden of malaria. Drug resistance complicates treatment, and it is one of the most important problems in malaria control. This study assessed the level of mutations in P. falciparum genes, pfdhfr, pfdhps, pfmdr1, and pfcrt, related to resistance to different anti-malarial drugs, in the Continental Region of Equatorial Guinea, after 8 years of implementing artesunate combination therapies as the first-line treatment. A triple mutant of pfdhfr (51I/59R/108N), which conferred resistance to sulfadoxine/pyrimethamine (SP), was found in 78% of samples from rural settings; its frequency was significantly different between urban and rural settings (p = 0.007). The 164L mutation was detected for the first time in this area, in rural settings (1.4%). We also identified three classes of previously described mutants and their frequencies: the partially resistant (pfdhfr 51I/59R/108N + pfdhps 437G), found at 54% (95% CI 47.75–60.25); the fully resistant (pfdhfr 51I/59R/108N + pfdhps 437G/540E), found at 28% (95% CI 7.07–14.93); and the super resistant (pfdhfr 51I/59R/108N + pfdhps 437G/540E/581G), found at 6% (95% CI 0.48–4.32). A double mutation in pfmdr1 (86Y + 1246Y) was detected at 2% (95% CI 0.24–3.76) frequency, distributed in both urban and rural samples. A combination of single mutations in the pfmdr1 and pfcrt genes (86Y + 76T), which was related to resistance to chloroquine and amodiaquine, was detected in 22% (95% CI 16.8–27.2) of samples from the area. The high level of mutations detected in P. falciparum genes related to SP resistance could be linked to the unsuccessful withdrawal of SP treatment in this area. Drug resistance can reduce the efficacy of intermittent prophylactic treatment with SP for children under 5 years old and for pregnant women. Although a high number of mutations was detected, the efficacy of the first-line treatment, artemisinin/amodiaquine, was not affected. To avoid increases in the numbers, occurrence, and spread of mutations, and to protect the population, the Ministry of Health should ensure that health centres and hospitals are supplied with appropriate first-line treatments for malaria.

Approximately 50 million people (60 %) live in malaria risk areas in Ethiopia, at altitudes below 2000 m. According to official data, 60–70 % of malaria cases are due to Plasmodium falciparum, and 40–30 % by Plasmodium vivax. The species Plasmodium ovale was detected in 2013 in the northwest of the country, being the first report of the presence of this species in Ethiopia since the 60 s. The aim of this study was to assess the diagnosis by microscopy and PCR, and demonstrate the presence of other Plasmodium species in the country. The survey was conducted in Bulbula, situated in the Rift Valley (West Arsi Province, Oromia Region). From December 2010 to October 2011, 3060 samples were collected from patients with symptoms of malaria; the diagnosis of malaria was done by microscopy and confirmation by PCR. 736 samples were positive for malaria by microscopy. After removing the 260 samples (109 positives and 151 negatives) for which it was not possible to do PCR, there were a total of 2800 samples, 1209 are used for its confirmation by PCR and quality control (627 are positives and 582 negatives by microscopy). From the 627 positive samples, 604 were confirmed as positive by PCR, 23 false positives were detected, and the group of 582 negative samples, 184 were positive by PCR (false negatives), which added to the previous positive samples is a total of 788, positive samples for some species of Plasmodium sp. 13.3 % more positives were detected with the PCR than the microscopy. Importantly, 23 samples were detected by PCR as P. ovale, after the sequencing of these samples was determined as P. ovale curtisi. The PCR detected more positive samples than the microscopy; in addition, P. ovale and P. ovale/P. vivax were detected that had not been detected by microscopy, which can affect in the infection control.