Isothermal nucleic acid amplification assays such as the loop mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to amplify the DNA. To further facilitate the use of LAMP assays in remote settings, simpler sample preparation methods and lyophilized reagents are required. The performance of a commercial malaria LAMP assay (Illumigene Malaria LAMP) was evaluated using two sample preparation workflows (simple filtration prep (SFP)) and gravity-driven filtration prep (GFP)) and pre-dispensed lyophilized reagents. Laboratory and clinical samples were tested in a field laboratory in Senegal and the results independently confirmed in a reference laboratory in the U.S.A. The Illumigene Malaria LAMP assay was easily implemented in the clinical laboratory and gave similar results to a real-time PCR reference test with limits of detection of ≤2.0 parasites/μl depending on the sample preparation method used. This assay reliably detected Plasmodium sp. parasites in a simple low-tech format, providing a much needed alternative to the more complex molecular tests for malaria diagnosis.

BackgroundMalaria in Nigeria is principally due to Plasmodium falciparum and, to a lesser extent to Plasmodium malariae and Plasmodium ovale. Plasmodium vivax is thought to be absent in Nigeria in particular and sub-Saharan Africa in general, due to the near fixation of the Duffy negative gene in this population. Nevertheless, there are frequent reports of P. vivax infection in Duffy negative individuals in the sub-region, including reports from two countries sharing border with Nigeria to the west (Republic of Benin) and east (Cameroon). Additionally, there were two cases of microscopic vivax-like malaria from Nigerian indigenous population. Hence molecular surveillance of the circulating Plasmodium species in two states (Lagos and Edo) of southwestern Nigeria was carried out.MethodsA cross-sectional survey between September 2016 and March 2017 was conducted. 436 febrile patients were included for the present work. Venous blood of these patients was subjected to RDT as well as microscopy. Further, parasite DNA was isolated from positive samples and PCR diagnostic was employed followed by direct sequencing of the 18S rRNA of Plasmodium species as well as sequencing of a portion of the promoter region of the Duffy antigen receptor for chemokines. Samples positive for P. vivax were re-amplified several times and finally using the High Fidelity Taq to rule out any bias introduced.ResultsOf the 256 (58.7%) amplifiable malaria parasite DNA, P. falciparum was, as expected, the major cause of infection, either alone 85.5% (219/256; 97 from Edo and 122 from Lagos), or mixed with P. malariae 6.3% (16/256) or with P. vivax 1.6% (4/256). Only one of the five P. vivax isolates was found to be a single infection. DNA sequencing and subsequent alignment of the 18S rRNA of P. vivax with the reference strains displayed very high similarities (100%). Remarkably, the T-33C was identified in all P. vivax samples, thus confirming that all vivax-infected patients in the current study are Duffy negative.ConclusionThe present study gave the first molecular evidence of P. vivax in Nigeria in Duffy negative individuals. Though restricted to two states; Edo in South–South and Lagos in South-west Nigeria, the real burden of this species in Nigeria and sub-Saharan Africa might have been underestimated, hence there is need to put in place a country-wide, as well as a sub-Saharan Africa-wide surveillance and appropriate control measures.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2588-7) contains supplementary material, which is available to authorized users.

The emergence of Plasmodium falciparum resistance to artemisinin in Southeast Asia threatens malaria control and elimination activities worldwide. Multiple polymorphisms in the P. falciparum kelch gene found in chromosome 13 (Pfk13) have been associated with artemisinin resistance. Surveillance of potential drug resistance loci within a population that may emerge under increasing drug pressure is an important public health activity. In this context, P. falciparum infections from an observational surveillance study in Senegal were genotyped using targeted amplicon deep sequencing (TADS) for Pfk13 polymorphisms. The results were compared to previously reported Pfk13 polymorphisms from around the world. A total of 22 Pfk13 propeller domain polymorphisms were identified in this study, of which 12 have previously not been reported. Interestingly, of the 10 polymorphisms identified in the present study that were also previously reported, all had a different amino acid substitution at these codon positions. Most of the polymorphisms were present at low frequencies and were confined to single isolates, suggesting they are likely transient polymorphisms that are part of naturally evolving parasite populations. The results of this study underscore the need to identify potential drug resistance loci existing within a population, which may emerge under increasing drug pressure.

Background The use of artemisinin as a monotherapy resulted in the emergence of artemisinin resistance in 2005 in Southeast Asia. Monitoring of artemisinin combination therapy (ACT) is critical in order to detect and prevent the spread of resistance in endemic areas. Ex vivo studies and genotyping of molecular markers of resistance can be used as part of this routine monitoring strategy. One gene that has been associated in some ACT partner drug resistance is the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene. The purpose of this study was to assess the drug susceptibility of P. falciparum populations from Thiès, Senegal by ex vivo assay and typing molecular markers of resistance to drug components of ACT currently used for treatment.Methods The ex vivo susceptibility of 170 P. falciparum isolates to chloroquine, amodiaquine, lumefantrine, artesunate, and artemether was determined using the DAPI ex vivo assay. The high resolution melting technique was used to genotype the pfmdr1 gene at codons 86, 184 and 1246.ResultsA significant decrease in IC50 values was observed between 2012 and 2013: from 13.84 to 6.484 for amodiaquine, 173.4 to 113.2 for lumefantrine, and 39.72 to 18.29 for chloroquine, respectively. Increase of the wild haplotype NYD and the decrease of the mutant haplotype NFD (79 and 62.26 %) was also observed. A correlation was observed between the wild type allele Y184 in pfmdr1 and higher IC50 for all drugs, except amodiaquine.ConclusionThis study has shown an increase in sensitivity over the span of two transmission seasons, marked by an increase in the WT alleles at pfmdr1. Continuous the monitoring of the ACT used for treatment of uncomplicated malaria will be helpful.

BackgroundMalaria transmission in Senegal is highly stratified, from low in the dry north to moderately high in the moist south. In northern Senegal, along the Senegal River Valley and in the Ferlo semi-desert region, annual incidence is less than five cases per 1000 inhabitants. Many nomadic pastoralists have permanent dwellings in the Ferlo Desert and Senegal River Valley, but spend dry season in the south with their herds, returning north when the rains start, leading to a concern that this population could contribute to ongoing transmission in the north.MethodsA modified snowball sampling survey was conducted at six sites in northern Senegal to determine the malaria prevention and treatment seeking practices and parasite prevalence among nomadic pastoralists in the Senegal River Valley and the Ferlo Desert. Nomadic pastoralists aged 6 months and older were surveyed during September and October 2014, and data regarding demographics, access to care and preventive measures were collected. Parasite infection was detected using rapid diagnostic tests (RDTs), microscopy (thin and thick smears) and polymerase chain reaction (PCR). Molecular barcodes were determined by high resolution melting (HRM).ResultsOf 1800 participants, 61% were male. Sixty-four percent had at least one bed net in the household, and 53% reported using a net the night before. Only 29% had received a net from a mass distribution campaign. Of the 8% (142) who reported having had fever in the last month, 55% sought care, 20% of whom received a diagnostic test, one-third of which (n = 5) were reported to be positive. Parasite prevalence was 0.44% by thick smear and 0.50% by PCR. None of the molecular barcodes identified among the nomadic pastoralists had been previously identified in Senegal.ConclusionsWhile access to and utilization of malaria control interventions among nomadic pastoralists was lower than the general population, parasite prevalence was lower than expected and sheds doubt on the perception that they are a source of ongoing transmission in the north. The National Malaria Control Program is making efforts to improve access to malaria prevention and case management for nomadic populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-2055-x) contains supplementary material, which is available to authorized users.

BackgroundExpanded malaria control efforts in Sénégal have resulted in increased use of rapid diagnostic tests (RDT) to identify the primary disease-causing Plasmodium species, Plasmodium falciparum. However, the type of RDT utilized in Sénégal does not detect other malaria-causing species such as Plasmodium ovale spp., Plasmodium malariae, or Plasmodium vivax. Consequently, there is a lack of information about the frequency and types of malaria infections occurring in Sénégal. This study set out to better determine whether species other than P. falciparum were evident among patients evaluated for possible malaria infection in Kédougou, Sénégal.MethodsReal-time polymerase chain reaction speciation assays for P. vivax, P. ovale spp., and P. malariae were developed and validated by sequencing and DNA extracted from 475 Plasmodium falciparum-specific HRP2-based RDT collected between 2013 and 2014 from a facility-based sample of symptomatic patients from two health clinics in Kédougou, a hyper-endemic region in southeastern Sénégal, were analysed.Results Plasmodium malariae (n = 3) and P. ovale wallikeri (n = 2) were observed as co-infections with P. falciparum among patients with positive RDT results (n = 187), including one patient positive for all three species. Among 288 negative RDT samples, samples positive for P. falciparum (n = 24), P. ovale curtisi (n = 3), P. ovale wallikeri (n = 1), and P. malariae (n = 3) were identified, corresponding to a non-falciparum positivity rate of 2.5%.ConclusionsThese findings emphasize the limitations of the RDT used for malaria diagnosis and demonstrate that non-P. falciparum malaria infections occur in Sénégal. Current RDT used for routine clinical diagnosis do not necessarily provide an accurate reflection of malaria transmission in Kédougou, Sénégal, and more sensitive and specific methods are required for diagnosis and patient care, as well as surveillance and elimination activities. These findings have implications for other malaria endemic settings where species besides P. falciparum may be transmitted and overlooked by control or elimination activities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1661-3) contains supplementary material, which is available to authorized users.

BackgroundMalaria treatment efforts are hindered by the rapid emergence and spread of drug resistant parasites. Simple assays to monitor parasite drug response in direct patient samples (ex vivo) can detect drug resistance before it becomes clinically apparent, and can inform changes in treatment policy to prevent the spread of resistance.MethodsParasite drug responses to amodiaquine, artemisinin, chloroquine and mefloquine were tested in approximately 400 Plasmodium falciparum malaria infections in Thiès, Senegal between 2008 and 2011 using a DAPI-based ex vivo drug resistance assay. Drug resistance-associated mutations were also genotyped in pfcrt and pfmdr1.ResultsParasite drug responses changed between 2008 and 2011, as parasites became less sensitive to amodiaquine, artemisinin and chloroquine over time. The prevalence of known resistance-associated mutations also changed over time. Decreased amodiaquine sensitivity was associated with sustained, highly prevalent mutations in pfcrt, and one mutation in pfmdr1 – Y184F – was associated with decreased parasite sensitivity to artemisinin.ConclusionsDirectly measuring ex vivo parasite drug response and resistance mutation genotyping over time are useful tools for monitoring parasite drug responses in field samples. Furthermore, these data suggest that the use of amodiaquine and artemisinin derivatives in combination therapies is selecting for increased drug tolerance within this population.

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