Seasonal malaria chemoprevention in a context of high presumed sulfadoxine-pyrimethamine resistance: malaria morbidity and molecular drug resistance profiles in South Sudan.

BACKGROUND: Seasonal malaria chemoprevention (SMC) using sulfadoxine-pyrimethamine plus amodiaquine (SP-AQ), is a community-based malaria preventive strategy commonly used in the Sahel region of sub-Saharan Africa. However, to date it has not been implemented in East Africa due to high SP resistance levels. This paper is a report on the implementation of SMC outside of the Sahel in an environment with a high level of presumed SP-resistance: five cycles of SMC using SPAQ were administered to children 3-59 months during a period of high malaria transmission (July-December 2019) in 21 villages in South Sudan. METHODS: A population-based SMC coverage survey was combined with a longitudinal time series analysis of health facility and community health data measured after each SMC cycle. SMC campaign effectiveness was assessed by Poisson model. SPAQ molecular resistance markers were additionally analysed from dried blood spots from malaria confirmed patients. RESULTS: Incidence of uncomplicated malaria was reduced from 6.6 per 100 to an average of 3.2 per 100 after SMC administration (mean reduction: 53%) and incidence of severe malaria showed a reduction from 21 per 10,000 before SMC campaign to a mean of 3.3 per 10,000 after each cycle (mean reduction: 84%) in the target group when compared to before the SMC campaign. The most prevalent molecular haplotype associated with SP resistance was the IRNGE haplotype (quintuple mutant, with 51I/59R/108N mutation in pfdhfr + 437G/540E in pfdhps). In contrast, there was a low frequency of AQ resistance markers and haplotypes resistant to both drugs combined (< 2%). CONCLUSIONS: The SMC campaign was effective and could be used as an additional preventive tool in seasonal malaria settings outside of the Sahel, especially in areas where access to health care is unstable. Malaria case load reduction was observed despite the high level of resistance to SP.

Screening for K13-Propeller Mutations Associated with Artemisinin Resistance in Plasmodium falciparum in Yambio County (Western Equatoria State, South Sudan).

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.

Deletion patterns, genetic variability and protein structure of pfhrp2 and pfhrp3: implications for malaria rapid diagnostic test in Amhara region, Ethiopia.

BACKGROUND: Although rapid diagnostic tests (RDTs) play a key role in malaria-control strategies, their efficacy has been threatened by deletion and genetic variability of the genes pfhrp2/3. This study aims to characterize the deletion, genetic patterns and diversity of these genes and their implication for malaria RDT effectiveness, as well as their genetic evolution in the Amhara region of Ethiopia. METHODS: The study included 354 isolates from symptomatic patients from the Amhara region of Ethiopia who tested positive by microscopy. Exon 1-2 and exon 2 of genes pfhrp2 and -3 were amplified, and exon 2 was sequenced to analyse the genetic diversity, phylogenetic relationship and epitope availability. RESULTS: The deletion frequency in exon 1-2 and exon 2 was 22 and 4.6% for pfhrp2, and 68 and 18% for pfhrp3, respectively. Double deletion frequency for pfhrp2 and pfhrp3 was 1.4%. High genetic diversity, lack of clustering by phylogenetic analysis and evidence of positive selection suggested a diversifying selection for both genes. The amino-acid sequences, classified into different haplotypes, varied widely in terms of frequency of repeats, with novel amino-acid changes. Aminoacidic repetition type 2 and type 7 were the most frequent in all the sequences. The most frequent epitopes among protein sequences were those recognized by MAbs 3A4 and C1-13. CONCLUSION: Deletions and high amino acidic variation in pfhrp2 and pfhrp3 suggest their possible impact on RDT use in the Amhara region, and the high genetic diversity of these genes could be associated with a diversifying selection in Ethiopia. Surveillance of these genes is, therefore, essential to ensure the effectiveness of public health interventions in this region.

Temporal evolution of the resistance genotypes of Plasmodium falciparum in isolates from Equatorial Guinea during 20 years (1999 to 2019).

BACKGROUND: Malaria is one of the deadliest diseases in the world, particularly in Africa. As such, resistance to anti-malarial drugs is one of the most important problems in terms of global malaria control. This study assesses the evolution of the different resistance markers over time and the possible influence of interventions and treatment changes that have been made in Equatorial Guinea. METHODS: A total of 1223 biological samples obtained in the period 1999 to 2019 were included in the study. Screening for mutations in the pfdhfr, pfdhps, pfmdr1, and pfcrt genes was carried out by nested PCR and restriction-fragment length polymorphisms (RFLPs), and the study of pfk13 genes was carried out by nested PCR, followed by sequencing to determine the presence of mutations. RESULTS: The partially and fully resistant haplotypes (pfdhfr + pfdhps) were found to increase over time. Moreover, in 2019, the fully resistant haplotype was found to be increasing, although its super-resistant counterpart remains much less prevalent. A continued decline in pfmdr1 and pfcrt gene mutations over time was also found. The number of mutations detected in pfk13 has increased since 2008, when artemisinin-based combination therapy (ACT) were first introduced, with more mutations being observed in 2019, with two synonymous and five non-synonymous mutations being detected, although these are not related to resistance to ACT. In addition, the non-synonymous A578S mutation, which is the most frequent on the African continent, was detected in 2013, although not in the following years. CONCLUSIONS: Withdrawal of the use of chloroquine (CQ) as a treatment in Equatorial Guinea has been shown to be effective over time, as wild-type parasite populations outnumber mutant populations. The upward trend observed in sulfadoxine-pyrimethamine (SP) resistance markers suggest its misuse, either alone or in combination with artesunate (AS) or amodiaquine (AQ), in some areas of the country, as was found in a previous study conducted by this group, which allows selective pressure from SP to continue. Single nucleotide polymorphisms (SNPs) 540E and 581G do not exceed the limit of 50 and 10%, respectively, thus meaning that SP is still effective as an intermittent preventive treatment (IPT) in this country. As for the pfk13 gene, no mutations have been detected in relation to resistance to ACT. However, in 2019 there is a greater accumulation of non-synonymous mutations compared to years prior to 2008.

First evidence of the deletion in the pfhrp2 and pfhrp3 genes in Plasmodium falciparum from Equatorial Guinea.

BACKGROUND: The World Health Organization (WHO) recommends rapid diagnostic tests (RDTs) as a good alternative malaria-diagnosis method in remote parts of sub-Saharan Africa. The majority of commercial RDTs currently available detect the Plasmodium falciparum protein histidine-rich protein 2 (PfHRP2). There have also been recent reports of pfhrp2 gene deletions being found in parasites collected from several African countries. The WHO has concluded that lacking the pfhrp2 gene must be monitored in Africa. The purpose of the study was to analyse why the samples that were positive by PCR were negative by RDTs and, therefore, to determine whether there have been deletions in the pfhrp2 and/or pfhrp3 genes. METHODS: Malaria NM-PCR was carried out on all the samples collected in the field. A group of 128 samples was positive by PCR but negative by RDT; these samples were classified as RDT false-negatives. PCR was carried out for exon2 of pfhrp2 and pfhrp3 genes to detect the presence or absence of these two genes. Frequencies with 95% confidence intervals (CIs) were used for prevalence estimates. Associations were assessed by the Chi square test or Fisher´s exact test. The level of significance was set at p ≤ 0.05. Statistical analyses were performed using the software package SPSSv.15.0. RESULTS: After PCR, 81 samples were identified (4.7%, 95% CI 3.8-5.8) which had deletion in both genes, pfhrp2 and pfhrp3. Overall, however, 11 samples (0.6%, 95% CI 0.36-1.14) had deletion only in pfhrp2 but not in pfhrp3, and 15 (0.9%, 95% CI 0.6-1.5) presented with deletion only in pfhrp3 but not in pfhrp2. Considering the pfhrp2 gene separately, within the total of 1724 samples, 92 (5.3%, 95% CI 4.37-6.5) had evidence of deletion. CONCLUSION: The present study provides the first evidence of deletion in the pfhrp2 and pfhrp3 genes in P. falciparum isolates from Equatorial Guinea. However, larger studies across different regions within the country and across different seasonal profiles are needed to determine the full extent of pfhrp2 and pfhrp3 deletion. It is strongly recommended to implement an active surveillance programme in order to detect any increases in pfhrp2 and pfhrp3 deletion frequencies.

Comparison of three diagnostic methods (microscopy, RDT, and PCR) for the detection of malaria parasites in representative samples from Equatorial Guinea.

BACKGROUND: Malaria in Equatorial Guinea remains a major public health problem. The country is a holo-endemic area with a year-round transmission pattern. In 2016, the prevalence of malaria was 12.09% and malaria caused 15% of deaths among children under 5 years. In the Continental Region, 95.2% of malaria infections were Plasmodium falciparum, 9.5% Plasmodium vivax, and eight cases mixed infection in 2011. The main strategy for malaria control is quick and accurate diagnosis followed by effective treatment. Early and accurate diagnosis of malaria is essential for both effective disease management and malaria surveillance. The quality of malaria diagnosis is important in all settings, as misdiagnosis can result in significant morbidity and mortality. Microscopy and RDTs are the primary choices for diagnosing malaria in the field. However, false-negative results may delay treatment and increase the number of persons capable of infecting mosquitoes in the community. The present study analysed the performance of microscopy and RDTs, the two main techniques used in Equatorial Guinea for the diagnosis of malaria, compared to semi-nested multiplex PCR (SnM-PCR). RESULTS: A total of 1724 samples tested by microscopy, RDT, and SnM-PCR were analysed. Among the negative samples detected by microscopy, 335 (19.4%) were false negatives. On the other hand, the negative samples detected by RDT, 128 (13.3%) were false negatives based on PCR. This finding is important, especially since it is a group of patients who did not receive antimalarial treatment. CONCLUSIONS: Owing to the high number of false negatives in microscopy, it is necessary to reinforce training in microscopy, the "Gold Standard" in endemic areas. A network of reference centres could potentially support ongoing diagnostic and control efforts made by malaria control programmes in the long term, as the National Centre of Tropical Medicine currently supports the National Programme against Malaria of Equatorial Guinea to perform all of the molecular studies necessary for disease control. Taking into account the results obtained with the RDTs, an exhaustive study of the deletion of the hrp2 gene must be done in EG to help choose the correct RDT for this area.

Profile of molecular mutations in pfdhfr, pfdhps, pfmdr1, and pfcrt genes of Plasmodium falciparum related to resistance to different anti-malarial drugs in the Bata District (Equatorial Guinea).

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Field Evaluation of Malaria Microscopy, Rapid Malaria Tests and Loop-Mediated Isothermal Amplification in a Rural Hospital in South Western Ethiopia.

BACKGROUND: In up to one third of the hospitals in some rural areas of Africa, laboratory services in malaria diagnosis are limited to microscopy by thin film, as no capability to perform thick film exists (gold standard in terms of sensitivity for malaria diagnosis). A new rapid molecular malaria diagnostic test called Loop-mediated isothermal DNA amplification (LAMP) has been recently validated in clinical trials showing exceptional sensitivity and specificity features. It could be a reliable diagnostic tool to be implemented without special equipment or training. OBJECTIVE: The objective of this proof of concept study was to confirm the feasibility of using LAMP technique for diagnosis of malaria in a rural Ethiopian hospital with limited resources. METHODOLOGY/PRINCIPAL FINDINGS: This study was carried out in Gambo General Hospital, West Arsi Province (Ethiopia), from November 1st to December 31st 2013. A total of 162 patients with a non-focal febrile syndrome were investigated. The diagnostic capability (sensitivity, specificity, positive predictive and negative predictive values) of rapid malaria tests and microscopy by thin film was evaluated in comparison with LAMP. Eleven (6.79%) out of the 162 patients with fever and suspected malaria, tested positive for LAMP, 3 (1.85%) for rapid malaria tests and none of the eleven cases was detected by thin film microscopy. CONCLUSIONS/SIGNIFICANCE: LAMP can be performed in basic rural laboratories without the need for specialized infrastructure and it may set a reliable tool for malaria control to detect a low level parasitemia.

Genetic diversity and signatures of selection of drug resistance in Plasmodium populations from both human and mosquito hosts in continental Equatorial Guinea.

BACKGROUND: In Plasmodium, the high level of genetic diversity and the interactions established by co-infecting parasite populations within the same host may be a source of selection on pathogen virulence and drug resistance. As different patterns have already been described in humans and mosquitoes, parasite diversity and population structure should be studied in both hosts to properly assess their effects on infection and transmission dynamics. This study aimed to characterize the circulating populations of Plasmodium spp and Plasmodium falciparum from a combined set of human blood and mosquito samples gathered in mainland Equatorial Guinea. Further, the origin and evolution of anti-malarial resistance in this area, where malaria remains a major public health problem were traced. METHODS: Plasmodium species infecting humans and mosquitoes were identified by nested-PCR of chelex-extracted DNA from dried blood spot samples and mosquitoes. Analysis of Pfmsp2 gene, anti-malarial-resistance associated genes, Pfdhps, Pfdhfr, Pfcrt and Pfmdr1, neutral microsatellites (STR) loci and Pfdhfr and Pfdhps flanking STR was undertaken to evaluate P. falciparum diversity. RESULTS: Prevalence of infection remains high in mainland Equatorial Guinea. No differences in parasite formula or significant genetic differentiation were seen in the parasite populations in both human and mosquito samples. Point mutations in all genes associated with anti-malarial resistance were highly prevalent. A high prevalence was observed for the Pfdhfr triple mutant in particular, associated with pyrimethamine resistance.Analysis of Pfdhps and Pfdhfr flanking STR revealed a decrease in the genetic diversity. This finding along with multiple independent introductions of Pfdhps mutant haplotypes suggest a soft selective sweep and an increased differentiation at Pfdhfr flanking microsatellites hints a model of positive directional selection for this gene. CONCLUSIONS: Chloroquine is no longer recommended for malaria treatment in Equatorial Guinea but sulphadoxine-pyrimethamine (SP) remains in use in combination with artesunate and is the only drug recommended in preventive chemotherapy in pregnancy. The high prevalence of point mutations in Pfdhfr and Pfdhps points to the danger of an eventual reduction in the efficacy of SP combined therapy in P. falciparum populations in Equatorial Guinea and to the essential continuous monitoring of these two genes.

Detection of high levels of mutations involved in anti-malarial drug resistance in Plasmodium falciparum and Plasmodium vivax at a rural hospital in southern Ethiopia.

BACKGROUND: In Ethiopia, malaria is caused by Plasmodium falciparum and Plasmodium vivax, and anti-malarial drug resistance is the most pressing problem confronting control of the disease. Since co-infection by both species of parasite is common and sulphadoxine-pyrimethamine (SP) has been intensively used, resistance to these drugs has appeared in both P. falciparum and P. vivax populations. This study was conducted to assess the prevalence of anti-malarial drug resistance in P. falciparum and P. vivax isolates collected at a rural hospital in southern Ethiopia. METHODS: A total of 1,147 patients with suspected malaria were studied in different months across the period 2007-2009. Plasmodium falciparum dhfr and dhps mutations and P. vivax dhfr polymorphisms associated with resistance to SP, as well as P. falciparum pfcrt and pfmdr1 mutations conferring chloroquine resistance, were assessed. RESULTS: PCR-based diagnosis showed that 125 of the 1147 patients had malaria. Of these, 52.8% and 37.6% of cases were due to P. falciparum and P. vivax respectively. A total of 10 cases (8%) showed co-infection by both species and two cases (1.6%) were infected by Plasmodium ovale. Pfdhfr triple mutation and pfdhfr/pfdhps quintuple mutation occurred in 90.8% (95% confidence interval [CI]: 82.2%-95.5%) and 82.9% (95% CI: 72.9%-89.7%) of P. falciparum isolates, respectively. Pfcrt T76 was observed in all cases and pfmdr1 Y86 and pfmdr1 Y1246 in 32.9% (95% CI: 23.4%-44.15%) and 17.1% (95% CI: 10.3-27.1%), respectively. The P. vivax dhfr core mutations, N117 and R58, were present in 98.2% (95% CI: 89.4-99.9%) and 91.2% (95% CI: 80.0-96.7%), respectively. CONCLUSION: Current molecular data show an extraordinarily high frequency of drug-resistance mutations in both P. falciparum and P. vivax in southern Ethiopia. Urgent surveillance of the emergence and spread of resistance is thus called for. The level of resistance indicates the need for implementation of entire population access to the new first-line treatment with artemether-lumefantrine, accompanied by government monitoring to prevent the emergence of resistance to this treatment.