Prevalence of mutations in the cysteine desulfurase IscS (Pfnfs1) gene in recurrent Plasmodium falciparum infections following artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) treatment in Matayos, Western Kenya.

BACKGROUND: Malaria remains a public health concern globally. Resistance to anti-malarial drugs has consistently threatened the gains in controlling the malaria parasites. Currently, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the treatment regimens against Plasmodium falciparum infections in many African countries, including Kenya. Recurrent infections have been reported in patients treated with AL or DP, suggesting the possibility of reinfection or parasite recrudescence associated with the development of resistance against the two therapies. The Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) K65 selection marker has previously been associated with decreased lumefantrine susceptibility. This study evaluated the frequency of the Pfnfs1 K65 resistance marker and associated K65Q resistant allele in recurrent infections collected from P. falciparum-infected individuals living in Matayos, Busia County, in western Kenya. METHODS: Archived dried blood spots (DBS) of patients with recurrent malaria infection on clinical follow-up days after treatment with either AL or DP were used in the study. After extraction of genomic DNA, PCR amplification and sequencing analysis were employed to determine the frequencies of the Pfnfs1 K65 resistance marker and K65Q mutant allele in the recurrent infections. Plasmodium falciparum msp1 and P. falciparum msp2 genetic markers were used to distinguish recrudescent infections from new infections. RESULTS: The K65 wild-type allele was detected at a frequency of 41% while the K65Q mutant allele was detected at a frequency of 22% in the recurrent samples. 58% of the samples containing the K65 wild-type allele were AL treated samples and while 42% were DP treated samples. 79% of the samples with the K65Q mutation were AL treated samples and 21% were DP treated samples. The K65 wild-type allele was detected in three recrudescent infections (100%) identified from the AL treated samples. The K65 wild-type allele was detected in two recrudescent DP treated samples (67%) while the K65Q mutant allele was identified in one DP treated (33%) recrudescent sample. CONCLUSIONS: The data demonstrate a higher frequency of the K65 resistance marker in patients with recurrent infection during the study period. The study underscores the need for consistent monitoring of molecular markers of resistance in regions of high malaria transmission.

Antimalarial activity assay of artesunate-3-chloro-4(4-chlorophenoxy) aniline in vitro and in mice models.

The global spread of multi-drug resistant P. falciparum, P. vivax, and P. malariae strains and absence of long-term effective vaccine makes chemotherapy the mainstay of malaria control strategies in endemic settings. The Mossman's assay and the Organization for Economic Co-operation and Development (OECD), 2001 guideline 423, were used to determine the cytotoxicity and acute oral toxicity of a novel hybrid drug, artesunate-3-Chloro-4(4-chlorophenoxy) aniline (ATSA), in vitro and in vivo, respectively. A modified Desjardins method was used to screen for antiplasmodial activity using P. falciparum (3D7 and W2) strains in vitro. The Peter's 4-day suppressive tests (4DTs) was used to evaluate the in vivo antimalaria activity using P. berghei ANKA strain, lumefantrine resistant (LuR), and piperaquine resistant (PQR) P. berghei lines. In silico prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles was assayed using PreADMET online prediction tool. The reference drug in all experiments was artesunate (ATS). Statistical significance between ATSA's activities in treated and control mice was evaluated by one-way analysis of variance (ANOVA). Results show that inhibitory concentrations-50 (IC50) of ATSA is 11.47 ± 1.3 (3D7) and 1.45 ± 0.26 (W2) against 4.66 ± 0.93 (3D7) and 0.60 ± 0.15 (W2) ng/ml of ATS with a selective index of 2180.91(3D7) and a therapeutic index (TI) of > 71). No mortalities were observed in acute oral toxicity assays and mean weight differences for test and controls were statistically insignificant (P > 0.05). The in vivo activity of ATSA was above 40% with effective dosage-50 (ED50) of 4.211, 2.601, and 3.875 mg/kg body weight against P. berghei ANKA, LuR, and PQR lines, respectively. The difference between treated and control mice was statistically significant (P < 0.05). ATSA has high intestinal absorption (HIA) > 95% and has medium human ether-a-go-go related gene (hERG) K+ channel inhibition risks. Preclinical and clinical studies on ATSA are recommended to evaluate its value in developing novel drugs for future management of multi-drug resistant malaria parasites.

Efficacy of artemisinin-lumefantrine for treatment of uncomplicated malaria after more than a decade of its use in Kenya.

BACKGROUND: The resistance of Plasmodium falciparum to antimalarial drugs remains a major impairment in the treatment and eradication of malaria globally. Following the introduction of artemisinin-based combination therapy (ACT), there have been reports of delayed parasite clearance. In Kenya, artemether-lumefantrine (AL) is the recommended first-line treatment of uncomplicated malaria. This study sought to assess the efficacy of AL after a decade of use as the preferred method of managing malarial infections in Kenya. We assessed clinical and parasitological responses of children under 5 years between May and November 2015 in Chulaimbo sub-County, Kisumu, Kenya. Patients aged between 6 and 60 months with uncomplicated P. falciparum mono-infection, confirmed through microscopy, were enrolled in the study. The patients were admitted at the facility for 3 days, treated with a standard dose of AL, and then put under observation for the next 28 days for the assessment of clinical and parasitological responses. Of the 90 patients enrolled, 14 were lost to follow-up while 76 were followed through to the end of the study period. Seventy-five patients (98.7%) cleared the parasitaemia within a period of 48 h while one patient (1.3%) cleared on day 3. There was 100% adequate clinical and parasitological response. All the patients cleared the parasites on day 3 and there were no re-infections observed during the stated follow-up period. This study, therefore, concludes that AL is highly efficacious in clearing P. falciparum parasites in children aged ≥6 and ≤60 months. The study, however, underscores the need for continued monitoring of the drug to forestall both gradual ineffectiveness and possible resistance to the drug in all target users.

Prevalence of pfdhfr and pfdhps mutations in Plasmodium falciparum associated with drug resistance among pregnant women receiving IPTp-SP at Msambweni County Referral Hospital, Kwale County, Kenya.

BACKGROUND: Prevention and treatment of malaria during pregnancy is crucial in dealing with maternal mortality and adverse fetal outcomes. The World Health Organization recommendation to treat all pregnant women with sulfadoxine-pyrimethamine (SP) through antenatal care structures was implemented in Kenya in the year 1998, but concerns about its effectiveness in preventing malaria in pregnancy has arisen due to the spread of SP resistant parasites. This study aimed to determine the prevalence of SP resistance markers in Plasmodium falciparum parasites isolated from pregnant women seeking antenatal care at Msambweni County Referral Hospital, located in coastal Kenya, between the year 2013 and 2015. METHODS: This hospital-based study included 106 malaria positive whole blood samples for analysis of SP resistance markers within the Pfdhfr gene (codons 51, 59 and 108) and Pfdhps gene (codons 437 and 540). The venous blood collected from all pregnant women was tested for malaria via light microscopy, then the malaria positive samples were separated into plasma and red cells and stored in a - 86° freezer for further studies. Archived red blood cells were processed for molecular characterization of SP resistance markers within the Pfdhfr and Pfdhps genes using real time PCR platform and Sanger sequencing. RESULTS: All samples had at least one mutation in the genes associated with drug resistance; polymorphism prevalence of Pfdhfr51I, 59R and 108N was at 88.7%, 78.3% and 93.4%, respectively, while Pfdhps polymorphism accounted for 94.3% and 91.5% at 437G and 540E, respectively. Quintuple mutations (at all the five codons) conferring total SP resistance had the highest prevalence of 85.8%. Quadruple mutations were observed at a frequency of 10.4%, and 24.5% had a mixed outcome of both wildtype and mutant genotypes in the genes of interest. CONCLUSION: The data suggest a high prevalence of P. falciparum genetic variations conferring resistance to SP among pregnant women, which may explain reduced efficacy of IPTp treatment in Kenya. There is need for extensive SP resistance profiling in Kenya to inform IPTp drug choices for successful malaria prevention during pregnancy.

Genetic diversity and population structure of Plasmodium falciparum in Kenyan-Ugandan border areas.

Kenya has, in the last decade, made tremendous progress in the fight against malaria. Nevertheless, continued surveillance of the genetic diversity and population structure of Plasmodium falciparum is required to refine malaria control and to adapt and improve elimination strategies. Twelve neutral microsatellite loci were genotyped in 201 P. falciparum isolates obtained from the Kenyan-Ugandan border (Busia) and from two inland malaria-endemic sites situated in western (Nyando) and coastal (Msambweni) Kenya. Analyses were done to assess the genetic diversity (allelic richness and expected heterozygosity, [He ]), multilocus linkage disequilibrium ( ISA ) and population structure. A similarly high degree of genetic diversity was observed among the three parasite populations surveyed (mean He  = 0.76; P > 0.05). Except in Msambweni, random association of microsatellite loci was observed, indicating high parasite out-breeding. Low to moderate genetic structure (FST  = 0.022-0.076; P < 0.0001) was observed with only 5% variance in allele frequencies observed among the populations. This study shows that the genetic diversity of P. falciparum populations at the Kenyan-Ugandan border is comparable to the parasite populations from inland Kenya. In addition, high genetic diversity, panmixia and weak population structure in this study highlight the fitness of Kenyan P. falciparum populations to successfully withstand malaria control interventions.

Le Kenya a réalisé d'énormes progrès au cours de la dernière décennie dans la lutte contre le paludisme. Néanmoins, une surveillance continue de la diversité génétique et de la structure de la population de P. falciparum est nécessaire pour affiner la lutte contre le paludisme et pour adapter et améliorer les stratégies d'élimination. Douze loci microsatellites neutres ont été génotypés chez 201 isolats de P. falciparum provenant de la frontière entre le Kenya et l'Ouganda (Busia) et de deux sites d'endémie palustre situés dans l'ouest (Nyando) et sur la côte (Msambweni), au Kenya. Des analyses ont été effectuées pour évaluer la diversité génétique (richesse allélique et hétérozygotie attendue, ([He]), déséquilibre de parenté des multiple loci ( ISA ) et structure de la population. Un degré hautement similaire de diversité génétique a été observé parmi les trois populations de parasites étudiées (He = 0,76; P > 0,05). A l'exception de Msambweni, une association aléatoire entre les microsatellites a été observée, indiquant une forte reproduction des parasites. Une structure génétique faible à modérée (FST  = 0,022-0,076; P < 0,0001) a été observée avec seulement 5% de variance dans la fréquence des allèles observée parmi les populations. Cette étude montre que la diversité génétique des populations de P. falciparum à la frontière entre le Kenya et l'Ouganda est comparable à celle des populations de parasites à l'intérieur du Kenya. De plus, la diversité génétique élevée, la panmixia et la structure démographique faible dans cette étude soulignent l'aptitude des populations de P. falciparum du Kenya à résister aux interventions de lutte contre le paludisme.

Gametocyte clearance in children, from western Kenya, with uncomplicated Plasmodium falciparum malaria after artemether-lumefantrine or dihydroartemisinin-piperaquine treatment.

BACKGROUND: The efficacy and safety of artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) against asexual parasites population has been documented. However, the effect of these anti-malarials on sexual parasites is still less clear. Gametocyte clearance following treatment is essential for malaria control and elimination efforts; therefore, the study sought to determine trends in gametocyte clearance after AL or DP treatment in children from a malaria-endemic site in Kenya. METHODS: Children aged between 0.5 and 12 years from Busia, western Kenya with uncomplicated Plasmodium falciparum malaria were assigned randomly to AL or DP treatment. A total of 334 children were enrolled, and dried blood spot samples were collected for up to 6 weeks after treatment during the peak malaria transmission season in 2016 and preserved. Plasmodium falciparum gametocytes were detected by qRT-PCR and gametocyte prevalence, density and mean duration of gametocyte carriage were determined. RESULTS: At baseline, all the 334 children had positive asexual parasites by microscopy, 12% (40/334) had detectable gametocyte by microscopy, and 83.7% (253/302) children had gametocytes by RT-qPCR. Gametocyte prevalence by RT-qPCR decreased from 85.1% (126/148) at day 0 to 7.04% (5/71) at day 42 in AL group and from 82.4% (127/154) at day 0 to 14.5% (11/74) at day 42 in DP group. The average duration of gametocyte carriage as estimated by qRT-PCR was slightly shorter in the AL group (4.5 days) than in the DP group (5.1 days) but not significantly different (p = 0.301). CONCLUSION: The study identifies no significant difference between AL and DP in gametocyte clearance. Gametocytes persisted up to 42 days post treatment in minority of individuals in both treatment arms. A gametocytocidal drug, in combination with artemisinin-based combination therapy, will be useful in blocking malaria transmission more efficiently.

Unclear association between levels of Plasmodium falciparum lactate dehydrogenase (PfLDH) in saliva of malaria patients and blood parasitaemia: diagnostic implications?

BACKGROUND: The use of saliva in diagnosis of infectious diseases is an attractive alternative to procedures that involve blood drawing. It promises to reduce risks associated with accidental needle pricks and improve patient compliance particularly in malaria survey and drug efficacy studies. Quantification of parasitaemia is useful in establishing severity of disease and in assessing individual patient response to treatment. In current practice, microscopy is the recommended technique, despite its limitations. This study measured the levels of Plasmodium falciparum lactate dehydrogenase (PfLDH) in saliva of malaria patients and investigated the relationship with blood parasitaemia. METHODS: Matched pre-treatment blood and saliva samples were collected from patients at Msambweni District Hospital, Kenya. Parasitaemia was determined and only those confirmed to be Plasmodium falciparum mono-infected were recruited. PfLDH was quantified in saliva using a commercial ELISA kit. A total of 175 samples were collected. Relationship between blood parasitaemia and concentration of PfLDH in saliva was determined using Pearson correlation statistics. F test was used to determine whether there is a significant difference between levels of PfLDH in saliva of patients with moderate to high parasitaemia and those with low parasitaemia. RESULTS: One-hundred and seventy-five patient samples were positive for malaria by microscopy. Of these, 62 (35%) tested positive for PfLDH in saliva, 113 (65%) were false negatives. For those that tested positive, (53) 85% were from patients with moderate to high parasitaemia while 9 (15%) were from patients with low parasitaemia. A correlation co-efficient of 0.18 indicated a weak positive relationship between the concentration of PfLDH in saliva and blood parasitaemia. There was a marginal difference between levels of PfLDH in saliva of patients with moderate to high parasitaemia and those with low parasitaemia [F (1, 59) = 1.83, p = 0.1807]. CONCLUSION: The results indicate that there is a weak correlation between levels of PfLDH in saliva and blood parasitaemia. This is weak association could be as a result of low sensitivity of the assay used as well as presence of inhibitors and proteases in saliva. Further studies should be focused towards reducing the number of false negatives and developing a customised assay that is specific for detection of PfLDH in saliva.

Efficacy and safety evaluation of a novel trioxaquine in the management of cerebral malaria in a mouse model.

BACKGROUND: The emergence of multidrug-resistant strains of Plasmodium falciparum poses a great threat of increased fatalities in cases of cerebral and other forms of severe malaria infections in which parenteral artesunate monotherapy is the current drug of choice. The study aimed to investigate in a mouse model of human cerebral malaria whether a trioxaquine chemically synthesized by covalent linking of a 4,7-dichloroquinoline pharmacophore to artesunate through a recent drug development approach termed 'covalent bitherapy' could improve the curative outcomes in cerebral malaria infections. METHODS: Human cerebral malaria rodent model, the C57BL/6 male mice were infected intraperitoneally (ip) with Plasmodium berghei ANKA and intravenously (iv) treated with the trioxaquine from day 8 post-infection (pi) at 12.5 and 25 mg/kg, respectively, twice a day for 3 days. Treatments with the trioxaquine precursors (artesunate and 4,7-dichloroquine), and quinine were also included as controls. In vivo safety evaluation for the trioxaquine was done according to Organization for Economic Co-operation and Development (OECD) guidelines 423, where female Swiss albino mice were orally administered with either 300 or 2000 mg/kg of the trioxaquine and monitored for signs of severity, and or mortality for 14 days post-treatment. RESULTS: The trioxaquine showed a potent and a rapid antiplasmodial activity with 80% parasite clearance in the first 24 h for the two dosages used. Long-term parasitaemia monitoring showed a total parasite clearance as the treated mice survived beyond 60 days post-treatment, with no recrudescence observed. Artesunate treated mice showed recrudescence 8 days post-treatment, with all mice in this group succumbing to the infection. Also, 4,7-dichloroquinoline and quinine did not show any significant parasitaemia suppression in the first 24 h post-treatment, with the animals succumbing to the infection. CONCLUSION: Covalent bitherapy proves to be a viable source of urgently needed new anti-malarials for management of cerebral malaria, and this polypharmacology approach could be a potential strategy to protect artesunate from parasite resistance and in potentially improving clinical outcomes in severe forms of malaria infections.

In vitro selection of Plasmodium falciparum Pfcrt and Pfmdr1 variants by artemisinin.

BACKGROUND: Anti-malarial drugs are the major focus in the prevention and treatment of malaria. Artemisinin-based combination therapy (ACT) is the WHO recommended first-line treatment for Plasmodium falciparum malaria across the endemic world. Also ACT is increasingly relied upon in treating Plasmodium vivax malaria where chloroquine is failing. The emergence of artemisinin drug-resistant parasites is a serious threat faced by global malaria control programmes. Therefore, the success of treatment and intervention strategies is highly pegged on understanding the genetic basis of resistance. METHODS: Here, resistance in P. falciparum was generated in vitro for artemisinin to produce levels above clinically relevant concentrations in vivo, and the molecular haplotypes investigated. Genomic DNA was extracted using the QIAamp mini DNA kit. DNA sequences of Pfk13, Pfcrt and Pfmdr1 genes were amplified by PCR and the amplicons were successfully sequenced. Single nucleotide polymorphisms were traced by standard bidirectional sequencing and reading the transcripts against wild-type sequences in Codon code Aligner Version 5.1 and NCBI blast. RESULTS: Exposure of parasite strains D6 and W2 to artemisinin resulted in a decrease in parasite susceptibility to artemisinin (W2 and D6) and lumefantrine (D6 only). The parasites exhibited elevated IC50s to multiple artemisinins, with >twofold resistance to artemisinin; however, the resistance index obtained with standard methods was noticeably less than expected for parasite lines recovered from 50 µg/ml 48 h drug pressure. The change in parasite susceptibility was associated with Pfmdr-185K mutation, a mutation never reported before. The Pfcrt-CVMNK genotype (Pfcrt codons 72-76) was retained and notably, the study did not detect any polymorphisms reported to reduce P. falciparum susceptibility in vivo in the coding sequences of the Pfk13 gene. DISCUSSION: This data demonstrate that P. falciparum has the capacity to develop resistance to artemisinin derivatives in vitro and that this phenotype is achieved by mutations in Pfmdr1, the genetic changes that are also underpinning lumefantrine resistance. This finding is of practical importance, because artemisinin drugs in Kenya are used in combination with lumefantrine for the treatment of malaria. CONCLUSION: Artemisinin resistance phenotype as has been shown in this work, is a decrease in parasites susceptibility to artemisinin derivatives together with the parasite's ability to recover from drug-induced dormancy after exposure to drug dosage above the in vivo clinical concentrations. The study surmises that Pfmdr1 may play a role in the anti-malarial activity of artemisinin.

Chloroquine sensitivity: diminished prevalence of chloroquine-resistant gene marker pfcrt-76 13 years after cessation of chloroquine use in Msambweni, Kenya.

BACKGROUND: Plasmodium falciparum resistance to chloroquine (CQ) denied healthcare providers access to a cheap and effective anti-malarial drug. Resistance has been proven to be due to point mutations on the parasite's pfcrt gene, particularly on codon 76, resulting in an amino acid change from lysine to threonine. This study sought to determine the prevalence of the pfcrt K76T mutation 13 years after CQ cessation in Msambweni, Kenya. METHODS: Finger-prick whole blood was collected on 3MM Whatman(®) filter paper from 99 falciparum malaria patients. Parasite DNA was extracted via the Chelex method from individual blood spots and used as template in nested PCR amplification of pfcrt. Apo1 restriction enzyme was used to digest the amplified DNA to identify the samples as wild type or sensitive at codon 76. Prevalence figures of the mutant pfcrt 76T gene were calculated by dividing the number of samples bearing the mutant gene with the total number of samples multiplied by 100 %. Chi square tests were used to test the significance of the findings against previous prevalence figures. RESULTS: Out of 99 clinical samples collected in 2013, prevalence of the mutant pfcrt 76T gene stood at 41 %. CONCLUSION: The results indicate a significant [χ(2) test, P ≤ 0.05 (2006 vs 2013)] reversal to sensitivity by the P. falciparum population in the study site compared to the situation reported in 2006 at the same study site. This could primarily be driven by diminished use of CQ in the study area in line with the official policy. Studies to establish prevalence of the pfcrt 76T gene could be expanded countrywide to establish the CQ sensitivity status and predict a date when CQ may be re-introduced as part of malaria chemotherapy.

Fitness cost of resistance for lumefantrine and piperaquine-resistant Plasmodium berghei in a mouse model.

BACKGROUND: The evolution of drug-resistant parasites is a major hindrance to malaria control, and thus understanding the behaviour of drug-resistant mutants is of clinical relevance. The study aimed to investigate how resistance against lumefantrine (LU) and piperaquine (PQ), anti-malarials used as partner drugs in artemisinin-based combination therapy (ACT), impacts parasite fitness. This is important since resistance to ACT, the first-line anti-malarial regimen is increasingly being reported. METHODS: The stability of Plasmodium berghei ANKA strain that was previously selected for LU and PQ resistance was evaluated using the 4-day assay and established infection test in mice. Fitness cost of resistance was determined by comparing parasites proliferation rates in absence of drug pressure for the drug-exposed parasites between day 4 and 7 post-infection (pi), relative to the wild-type. Statistical analysis of data to compare mean parasitaemia and growth rates of respective parasite lines was carried out using student's t-test and one-way analysis of variance, with significance level set at p<0.05. RESULTS: During serial passaging in the absence of the drug, the PQ-resistant parasite maintained low growth rates at day 7 pi (mean parasitaemia, 5.6% ± 2.3) relative to the wild-type (28.4% ± 6.6), translating into a fitness cost of resistance of 80.3%. Whilst resistance phenotype for PQ was stable, that of LU was transient since after several serial passages in the absence of drug, the LU-exposed line assumed the growth patterns of the wild-type. CONCLUSIONS: The contrasting behaviour of PQ- and LU-resistance phenotypes support similar findings which indicate that even for drugs within the same chemical class, resistance-conferred traits may vary on how they influence parasite fitness and virulence. Resistance-mediating polymorphisms have been associated with less fit malaria parasites. In the absence of drug pressure in the field, it is therefore likely that the wild-type parasite will out-compete the mutant form. This implies the possibility of reintroducing a drug previously lost to resistance, after a period of suspended use. Considering the recent reports of high failure rates associated with ACT, high fitness cost of resistance to PQ is therefore of clinical relevance as the drug is a partner in ACT.

Detection of mixed infection level of Plasmodium falciparum and Plasmodium vivax by SYBR Green I-based real-time PCR in North Gondar, north-west Ethiopia.

BACKGROUND: Malaria is caused by five Plasmodium species and transmitted by anopheline mosquitoes. It occurs in single and mixed infections. Mixed infection easily leads to misdiagnosis. Accurate detection of malaria species is vital. Therefore, the study was conducted to determine the level of mixed infection and misdiagnosis of malaria species in the study area using SYBR Green I-based real time PCR. METHODS: The study was conducted in seven health centres from North Gondar, north-west Ethiopia. The data of all febrile patients, who attended the outpatient department for malaria diagnosis, from October to December 2013, was recorded. Dried blood spots were prepared from 168 positive samples for molecular re-evaluation. Parasite DNA was extracted using a commercial kit and Plasmodium species were re-evaluated with SYBR Green I-based real time PCR to detect mixed infections and misdiagnosed mono-infections. RESULTS: Among 7343 patients who were diagnosed for malaria in six study sites within the second quarter of the Ethiopian fiscal year (2013) 1802 (24.54%) were positive for malaria parasite. Out of this, 1,216 (67.48%) Plasmodium falciparum, 553 (30.68%) Plasmodium vivax and 33 (1.8%) mixed infections of both species were recorded. The result showed high prevalence of P. falciparum and P. vivax, but very low prevalence of mixed infections. Among 168 samples collected on dried blood spot 7 (4.17%) were P. vivax, 158 (94.05%) were P. falciparum and 3 (1.80%) were mixed infections of both species. After re-evaluation 10 (5.95%) P. vivax, 112 (66.67%) P. falciparum, 21 (12.50%) P. falciparum + P. vivax mixed infection, and 17 (10.12%) Plasmodium ovale positive rate was recorded. The re-evaluation showed high level of mixed infection, and misdiagnosis of P. ovale and P. vivax. CONCLUSIONS: The result shows that P. falciparum prevalence is higher than P. vivax in the study area. The results, obtained from SYBR Green I-based real time PCR, indicated that the diagnosis efficiency of microscopy is very low for species-specific and mixed infection detection. Therefore, real time PCR-based species diagnosis should be applied for clinical diagnosis and quality control purposes in order to prevent the advent of drug resistant strains due to misdiagnosis and mistreatment.

Kenya's emergency-hire nursing programme: a pilot evaluation of health service delivery in two districts.

OBJECTIVE: To assess the feasibility of utilizing a small-scale, low-cost, pilot evaluation in assessing the short-term impact of Kenya's emergency-hire nursing programme (EHP) on the delivery of health services (outpatient visits and maternal-child health indicators) in two underserved health districts with high HIV/AIDS prevalence. METHODS: Six primary outcomes were assessed through the collection of data from facility-level health management forms-total general outpatient visits, vaginal deliveries, caesarean sections, antenatal care (ANC) attendance, ANC clients tested for HIV, and deliveries to HIV-positive women. Data on outcome measures were assessed both pre-and post-emergency-hire nurse placement. Informal discussions were also conducted to obtain supporting qualitative data. FINDINGS: The majority of EHP nurses were placed in Suba (15.5%) and Siaya (13%) districts. At the time of the intervention, we describe an increase in total general outpatient visits, vaginal deliveries and caesarean sections within both districts. Similar significant increases were seen with ANC attendance and deliveries to HIV-positive women. Despite increases in the quantity of health services immediately following nurse placement, these levels were often not sustained. We identify several factors that challenge the long-term sustainability of these staffing enhancements. CONCLUSIONS: There are multiple factors beyond increasing the supply of nurses that affect the delivery of health services. We believe this pilot evaluation sets the foundation for future, larger and more comprehensive studies further elaborating on the interface between interventions to alleviate nursing shortages and promote enhanced health service delivery. We also stress the importance of strong national and local relationships in conducting future studies.

Constituents of the roots and leaves of Ekebergia capensis and their potential antiplasmodial and cytotoxic activities.

A new triterpenoid, 3-oxo-12ß-hydroxy-oleanan-28,13ß-olide (1), and six known triterpenoids 2-7 were isolated from the root bark of Ekebergia capensis, an African medicinal plant. A limonoid 8 and two glycoflavonoids 9-10 were found in its leaves. The metabolites were identified by NMR and MS analyses, and their cytotoxicity was evaluated against the mammalian African monkey kidney (vero), mouse breast cancer (4T1), human larynx carcinoma (HEp2) and human breast cancer (MDA-MB-231) cell lines. Out of the isolates, oleanonic acid (2) showed the highest cytotoxicity, i.e., IC50's of 1.4 and 13.3 µM against the HEp2 and 4T1 cells, respectively. Motivated by the higher cytotoxicity of the crude bark extract as compared to the isolates, the interactions of oleanonic acid (2) with five triterpenoids 3-7 were evaluated on vero cells. In an antiplasmodial assay, seven of the metabolites were observed to possess moderate activity against the D6 and W2 strains of P. falciparum (IC50 27.1-97.1 µM), however with a low selectivity index (IC50(vero)/IC50(P. falciparum-D6)<10). The observed moderate antiplasmodial activity may be due to general cytotoxicity of the isolated triterpenoids.

Evaluation of the antimalarial properties of Solanum incanum L. leaf extract fractions and its ability to downregulate delta aminolevulinate dehydratase to prevent the establishment of malaria infection.

ETHNOPHARMACOLOGICAL RELEVANCE: Solanum incanum L. is commonly used in traditional herbal medicine (THM) in Kenya for treating various ailments. Recent developments in disease treatment have introduced the concept of host-directed therapy (HDT). This approach involves targeting factors within the host cell that can impede the growth or replication of a pathogen. One such host factor is delta aminolevulinate dehydratase (δ-ALAD), the second enzyme in the heme biosynthesis pathway utilized by Plasmodium for growth. Studies using mice models have shown an increase in δ-ALAD expression during Plasmodium berghei infection. Another plant in the Solanum genus, S. guaranticum, has been found to inhibit δ-ALAD in red blood cells in vitro and in the brain in vivo. Is it possible that the bioactive compounds in S. incanum extracts could also be effective in HDT for malaria treatment? AIM OF STUDY: To better assess the effectiveness of S. incanum leaf extracts as a curative and prophylaxis in malaria parasite infection, and to test the plant's ability to decrease δ-ALAD expression. MATERIALS AND METHODS: The leaves of S. incanum were collected, dried, and pulverized before being subjected to a successive extraction protocol to obtain crude, hexane, ethyl acetate, and aqueous extract fractions. Phytochemical analysis was conducted on all extract fractions, followed by GC-MS analysis of the fraction with the most potent antimalarial activity. An acute toxicity study was also performed on the extracted fractions. The potency of the extract fractions as curative and prophylactic antimalarial was then evaluated in THM using Plasmodium berghei-infected mice at a dose of 100 mg/kg. The extract fraction with the highest activity was further evaluated at varying doses and its effect on δ-ALAD was measured using RT-qPCR. The percentage of parasitemia and chemosuppression, and mean survival time were used as indices of activity. RESULTS: Phytochemical analysis revealed that the ethyl acetate and aqueous extract fractions contained high terpenoids, flavonoids, and phenols levels. However, alkaloids were only present in moderate quantities in the aqueous extract, and quinones were found in high levels only in the crude extract. Additionally, all extract fractions contained saponins in high levels but lacked tannins. While the plant extracts were found to be non-toxic, they did not exhibit curative antimalarial activity. However, all extract fractions showed prophylactic antimalarial activity, with the ethyl acetate extract having the highest percentage of chemosuppression even at doses of 250 and 1000 mg/kg. In the negative control, the expression of δ-ALAD was 5.4-fold, but this was significantly reduced to 2.3-fold when mice were treated with 250 mg/kg of the ethyl acetate fraction. GC-MS analysis of the ethyl acetate fraction revealed high percentages of 2-methyloctacosane, tetracosane, and decane. CONCLUSION: The fractions extracted from S. incanum leaves have been found to possess only antimalarial prophylactic properties, with the ethyl acetate extract fraction showing the most effective results. The activity of this fraction may be attributed to its ability to decrease the expression of δ-ALAD, as it contains an alkane compound implicated with enzyme-inhibitory activity.

Expression of the Fab enzymes (Fab I and Fab Z) from Plasmodium falciparum after exposure to Artemisia afra plant extracts and drugs screening.

The emergence and spread of drug resistance of the malaria parasite to the main treatment emphasize the need to develop new antimalarial drugs. In this context, the fatty acid biosynthesis (FAS_II) pathway of the malaria parasite is one of the ideal targets due to its crucial role in parasite survival. In this study, we report the expression and the affinity binding of Fab_I and Fab_Z after exposure to the parasite with different extracts of the Artemisia afra. The parasites were exposed for 2 days to different extracts. Gene expression was done to determine the level of expression of the fab enzymes after treatments. A GCMS was run to determine the different compounds of the plant extracts, followed by a virtual screening between the fab enzymes and the active compounds using Pyrex. The results showed different expression patterns of the Fab enzymes. Fab_I expression was downregulated in the W2 and D6 strains by the ethanolic extract but was increased by Hexane and DCM extracts. A different expression pattern was observed for Fab_Z. It was all upregulated except in the D6 strain when exposed to the ethanolic and hexane extracts. Virtual screening showed an affinity with many compounds. Hits compounds with high binding energy were detected. 11alphaHydroxyprogesterone and Aspidospermidin-17-ol were found to have high binding energy with Fab_I respectively (- 10.7 kcal/mol; - 10.2 kcal/mol). Fab_Z shows also high affinity with 11alpha-Hydroxyprogesterone (- 10 kcal/mol) and Thiourea (- 8.4 kcal/mol). This study shows the potential of A. afra to be used as a new source of novel antimalarial compounds.

In vivo antiplasmodial activities of stem bark extracts of Avicennia marina in Plasmodium berghei-infected mice.

Introduction: malaria remains the leading cause of morbidity and mortality in developing tropical and subtropical nations. Due to the emergence and spread of drug resistance to currently available drugs, there is a need for the search of novel, safe, and reasonably affordable anti-malarial medications. The objective of this study was to assess the in vivoanti-malarial effectiveness of Avicennia marina stem bark extracts in a mice model. Methods: guidelines 425 of the Organization for Economic Cooperation and Development were used to determine the extracts' acute toxicity. Mice infected with chloroquine-sensitive Plasmodium berghei (ANKA strain) were tested for in vivoanti-plasmodial activity, and by giving oral doses of 100 mg/kg, 250 mg/kg, and 500 mg/kg body weight of extracts, the plant's suppressive, curative, and preventive effects were assessed. Results: mice treated with dosages of up to 5000 mg/kg showed no evidence of acute toxicity or mortality. Consequently, it was determined that the acute lethal dosage of Avicennia marina extracts in swiss albino mice was greater than 5000 mg/kg. All doses of the extracts exhibited significant (p<0.05) dose-dependent suppression of P. berghei in the suppressive tests compared to the control group. At the highest dose (500 mg/kg), Methanolic crude extracts exerted the highest (93%) parasitemia suppression during the 4-day suppressive test. The extracts also displayed significant (p<0.001) prophylactic and curative activities at all doses compared to the control. Conclusion: results from this study ascertained the safety and promising curative, prophylactic and suppressive anti-plasmodial capabilities of the stem bark extracts of Avicennia marina in mice model.

Detection of Wuchereria bancrofti in human blood samples and mosquitoes in Matayos, Busia County-Kenya.

Lymphatic filariasis is a mosquito borne disease which leads to abnormal painful enlarged body parts, severe disability and social stigma. We screened Wuchereria bancrofti in Matayos constituency in Busia County. Blood samples were collected from 23 villages selected purposively based on clinical case reports. Finger prick and/or venous blood sampling and mosquito collections was carried out. Antigenaemia and filarial DNA prevalence were determined. Infection rates on mosquito pools were estimated and SPSS version 26 was used for descriptive statistics analysis. A total of 262 participants were recruited, 73.3% (n = 192) of the participants had no symptoms, 14.1% (n = 5.3) had swollen legs, 5.3% (n = 14) had painful legs and 3.8% (n = 10) with scrotal swellings. Average antigenemia prevalence was 35.9% (n = 94) and DNA prevalence was at 8.0% (n = 21). A total of 1305 mosquitoes were collected and pooled into 2-20 mosquitoes of the same species and from the same village. Two pools out of 78 were positive for filarial DNA with a minimum infection rate of 0.15%. From this study, antigenaemia and infected mosquitoes are an indication of active transmission. The clinical signs are evidence that filarial infections have been in circulation for over 10 years. The global climate change phenomenon currently happening has been shown to adversely affect the transmission of vector borne diseases and is likely to increase lymphatic filariasis transmission in the area. This study therefore recommends further screening before Mass Drug Administration, morbidity management and enhanced mosquito control Programmes are recommended in the study area.

Amodiaquine drug pressure selects nonsynonymous mutations in pantothenate kinase 1, diacylglycerol kinase, and phosphatidylinositol-4 kinase in Plasmodium berghei ANKA.

Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ), the long-acting components of the artemisinin-based combination therapies (ACTs), are a cornerstone of malaria treatment in Africa. Studies have shown that PQ, AQ, and LM resistance may arise independently of predicted modes of action. Protein kinases have emerged as mediators of drug action and efficacy in malaria parasites; however, the link between top druggable Plasmodium kinases with LM, PQ, and AQ resistance remains unclear. Using LM, PQ, or AQ-resistant Plasmodium berghei parasites, we have evaluated the association of choline kinase (CK), pantothenate kinase 1 (PANK1), diacylglycerol kinase (DAGK), and phosphatidylinositol-4 kinase (PI4Kß), and calcium-dependent protein kinase 1 (CDPK1) with LM, PQ, and AQ resistance in Plasmodium berghei ANKA. Methods: We used in silico bioinformatics tools to identify ligand-binding motifs, active sites, and sequence conservation across the different parasites. We then used PCR and sequencing analysis to probe for single nucleotide polymorphisms (SNPs) within the predicted functional motifs in the CK, PANK1, DAGK, PI4Kß, and CDPK1. Using qPCR analysis, we finally measured the mRNA amount of PANK1, DAGK, and PI4Kß at trophozoites and schizonts stages. Results: We reveal sequence conservation and unique ligand-binding motifs in the CK, PANK1, DAGK, PI4Kß, and CDPK1 across malaria species. DAGK, PANK1, and PI4Kß possessed nonsynonymous mutations; surprisingly, the mutations only occurred in the AQr parasites. PANK1 acquired Asn394His while DAGK contained K270R and K292R mutations. PI4Kß had Asp366Asn, Ser1367Arg, Tyr1394Asn and Asp1423Asn. We show downregulation of PANK1, DAGK, and PI4Kß in the trophozoites but upregulation at the schizonts stages in the AQr parasites. Conclusions: The selective acquisition of the mutations and the differential gene expression in AQ-resistant parasites may signify proteins under AQ pressure. The role of the mutations in the resistant parasites and the impact on drug responses require further investigations in malaria parasites.

Host candidate gene polymorphisms and clearance of drug-resistant Plasmodium falciparum parasites.

BACKGROUND: Resistance to anti-malarial drugs is a widespread problem for control programmes for this devastating disease. Molecular tests are available for many anti-malarial drugs and are useful tools for the surveillance of drug resistance. However, the correlation of treatment outcome and molecular tests with particular parasite markers is not perfect, due in part to individuals who are able to clear genotypically drug-resistant parasites. This study aimed to identify molecular markers in the human genome that correlate with the clearance of malaria parasites after drug treatment, despite the drug resistance profile of the protozoan as predicted by molecular approaches. METHODS: 3721 samples from five African countries, which were known to contain genotypically drug resistant parasites, were analysed. These parasites were collected from patients who subsequently failed to clear their infection following drug treatment, as expected, but also from patients who successfully cleared their infections with drug-resistant parasites. 67 human polymorphisms (SNPs) on 17 chromosomes were analysed using Sequenom's mass spectrometry iPLEX gold platform, to identify regions of the human genome, which contribute to enhanced clearance of drug resistant parasites. RESULTS: An analysis of all data from the five countries revealed significant associations between the phenotype of ability to clear drug-resistant Plasmodium falciparum infection and human immune response loci common to all populations. Overall, three SNPs showed a significant association with clearance of drug-resistant parasites with odds ratios of 0.76 for SNP rs2706384 (95% CI 0.71-0.92, P = 0.005), 0.66 for SNP rs1805015 (95% CI 0.45-0.97, P = 0.03), and 0.67 for SNP rs1128127 (95% CI 0.45-0.99, P = 0.05), after adjustment for possible confounding factors. The first two SNPs (rs2706384 and rs1805015) are within loci involved in pro-inflammatory (interferon-gamma) and anti-inflammatory (IL-4) cytokine responses. The third locus encodes a protein involved in the degradation of misfolded proteins within the endoplasmic reticulum, and its role, if any, in the clearance phenotype is unclear. CONCLUSIONS: The study showed significant association of three loci in the human genome with the ability of parasite to clear drug-resistant P. falciparum in samples taken from five countries distributed across sub-Saharan Africa. Both SNP rs2706384 and SNP1805015 have previously been reported to be associated with risk of malaria infection in African populations. The loci are involved in the Th1/Th2 balance, and the association of SNPs within these genes suggests a key role for antibody in the clearance of drug-resistant parasites. It is possible that patients able to clear drug-resistant infections have an enhanced ability to control parasite growth.