A new triterpenoid, 3-oxo-12β-hydroxy-oleanan-28,13β-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.

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. 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. Out of 99 clinical samples collected in 2013, prevalence of the mutant pfcrt 76T gene stood at 41 %. The results indicate a significant [χ2 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.

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. 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. 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. 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.

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. 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. 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. 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.

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.

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. 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. 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]. 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.