Yaye Die Ndiaye

Evaluation of the Illumigene Malaria LAMP: A Robust Molecular Diagnostic Tool for Malaria Parasites.

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.

Changes in drug sensitivity and anti-malarial drug resistance mutations over time among Plasmodium falciparum parasites in Senegal

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.

Polymorphism in dhfr/dhps genes, parasite density and ex vivo response to pyrimethamine in plasmodium falciparum malaria parasites in thies, senegal

Resistance to sulfadoxine-pyrimethamine (SP) in Plasmodium falciparum malaria parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes, and these mutations have spread resistance worldwide. SP, used for several years in Senegal, has been recommended for intermittent preventive treatment for malaria in pregnancy (IPTp) and has been widely implemented since 2003 in this country. There is currently limited data on SP resistance from molecular marker genotyping, and no data on pyrimethamine ex vivo sensitivity in Senegal. Molecular markers of SP resistance and pyrimethamine ex vivo sensitivity were investigated in 416 parasite samples collected from the general population, from the Thies region between 2003 and 2011. The prevalence of the N51I/C59R/S108N triple mutation in dhfr increased from 40% in 2003 to 93% in 2011. Furthermore, the prevalence of the dhfr N51I/C59R/S108N and dhps A437G quadruple mutation increased, from 20% to 66% over the same time frame, then down to 44% by 2011. There was a significant increase in the prevalence of the dhfr triple mutation, as well as an association between dhfr genotypes and pyrimethamine response. Conversely, dhps mutations in codons 436 and 437 did not show consistent variation between 2003 and 2011. These findings suggest that regular screening for molecular markers of antifolate resistance and ex vivo drug response monitoring should be incorporated with ongoing in vivo efficacy monitoring in areas where IPTp-SP is implemented and where pyrimethamine and sulfa drugs are still widely administered in the general population.

Molecular Epidemiology of Plasmodium falciparum kelch13 Mutations in Senegal Determined by Using Targeted Amplicon Deep Sequencing

ABSTRACT 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.

Tinea pedis due to Cylindrocarpon lichenicola beginning onycholysis.

A 33 year old woman presented with both feet, humid and white Tinea pedis at the second, third and fourth inter-toes areas associated with a beginning onycholysis of the nails lasting for 18 months. KOH mount of the samples was positive for fungal hyphae. The fungus was isolated on Sabouraud-chlorampphenicol agar and identified as Cylindrocarpon lichenicola. The patient was treated with an association of terbinafine tablet and terbinafine cream and presented clinical cure after three months.

High resolution melting: a useful field-deployable method to measure dhfr and dhps drug resistance in both highly and lowly endemic Plasmodium populations

BackgroundEmergence and spread of drug resistance to every anti-malarial used to date, creates an urgent need for development of sensitive, specific and field-deployable molecular tools for detection and surveillance of validated drug resistance markers. Such tools would allow early detection of mutations in resistance loci. The aim of this study was to compare common population signatures and drug resistance marker frequencies between two populations with different levels of malaria endemicity and history of anti-malarial drug use: Tanzania and Sénégal. This was accomplished by implementing a high resolution melting assay to study molecular markers of drug resistance as compared to polymerase chain reaction–restriction fragment length polymorphism (PCR/RFLP) methodology.MethodsFifty blood samples were collected each from a lowly malaria endemic site (Sénégal), and a highly malaria endemic site (Tanzania) from patients presenting with uncomplicated Plasmodium falciparum malaria at clinic. Data representing the DHFR were derived using both PCR–RFLP and HRM assay; while genotyping data representing the DHPS were evaluated in Senegal and Tanzania using HRM. Msp genotyping analysis was used to characterize the multiplicity of infection in both countries.ResultsA high prevalence of samples harbouring mutant DHFR alleles was observed in both population using both genotyping techniques. HRM was better able to detect mixed alleles compared to PCR/RFLP for DHFR codon 51 in Tanzania; and only HRM was able to detect mixed infections from Senegal. A high prevalence of mutant alleles in DHFR (codons 51, 59, 108) and DHPS (codon 437) were found among samples from Sénégal while no mutations were observed at DHPS codons 540 and 581, from both countries. Overall, the frequency of samples harbouring either a single DHFR mutation (S108N) or double mutation in DHFR (C59R/S108N) was greater in Sénégal compared to Tanzania.ConclusionHere the results demonstrate that HRM is a rapid, sensitive, and field-deployable alternative technique to PCR–RFLP genotyping that is useful in populations harbouring more than one parasite genome (polygenomic infections). In this study, a high levels of resistance polymorphisms was observed in both dhfr and dhps, among samples from Tanzania and Sénégal. A routine monitoring by molecular markers can be a way to detect emergence of resistance involving a change in the treatment policy.

Distribution of Parasites Detected in Stool Samples of Patients in Le Dantec University Hospital of Dakar, Senegal, from 2011 to 2015

To identify the parasites responsible for intestinal parasitic infections diagnosed at Le Dantec University Hospital of Dakar, distribution of parasites detected in stool samples of patients was studied. From 2011 to 2015, 2578 patients were included in the study. A direct examination and Ritchie technique were performed as parasite search techniques. In total, 408 samples were positive showing 440 intestinal parasites; this corresponds to prevalence of 15.8%. Parasites were detected in monoparasitism (85.7%) and multiparasitism (14.3%). The most common species found in monoparasitism were Entamoeba coli (38.9%), E. histolytica/dispar (12.7%), Giardia intestinalis (8%), and Ascaris lumbricoides (7.3%). The most common associations were A. lumbricoides-Trichuris trichiura (3.6%) and E. coli-G. intestinalis (2.7%). Nonhospitalized patients were significantly more affected with 65.4% compared to hospitalized counterparts; and also there were more men (50.7%) than women. With 67.4%, adults were the most affected age group, while the elderly were less affected with only 7% (p = 0.5). This study shows increasing prevalence of intestinal parasitic infections over the years. So health education should be promoted in addition to the already begun mass treatment program. This would help to limit or even halt the spread of these diseases.

Le paludisme à Plasmodium ovale wallikeri et Plasmodium ovale curtisi au Sénégal en 2016

Recently in Senegal, a case of Plasmodium ovale malaria had led to a diagnostic difficulty due to the ignorance of this parasite and the neglect of it. The objective of this study was to actively investigate cases of P. ovale malaria that would be misdiagnosed in the health centre structures of Senegal. The study was conducted in three areas that reflect different epidemiological strata of malaria. Microscopy was performed by microscopy experts on suspected malaria patients. The results were validated by Rougemont real-time PCR. Positive P. ovale cases were genotyped by nested PCR targeting the potra gene. A total of 406 samples were taken. Microscopy of Giemsa stained thick and thin smears recorded 228 cases of Plasmodium falciparum (97%), 3 cases of Plasmodium malariae (1.3%), and 4 cases of P. ovale (1.7%). The cases of P. ovale observed at microscopy were confirmed by real-time PCR. Genotyping of P. ovale revealed 3 cases of P. ovale wallikeri and 1 case of P. ovale curtisi. The prevalence of P. ovale malaria remains low in Senegal. However, malaria microscopists should be trained to recognize non-falciparum species in order to avoid the diagnostic delays and unnecessary investigations. National malaria control program should consider those species for the better management of malaria control in the country. Simplified molecular methods like, loop-mediated isothermal amplification (LAMP) may be useful to better characterize the epidemiology of non-falciparum malaria.