Kelch 13-propeller polymorphisms in Plasmodium falciparum from Jazan region, southwest Saudi Arabia.

BACKGROUND: Artemisinin-based combination therapy (ACT) is recommended at the initial phase for treatment of Plasmodium falciparum, to reduce morbidity and mortality in all countries where malaria is endemic. Polymorphism in portions of P. falciparum gene encoding kelch (K13)-propeller domains is associated with delayed parasite clearance after ACT. Of about 124 different non-synonymous mutations, 46 have been identified in Southeast Asia (SEA), 62 in sub-Saharan Africa (SSA) and 16 in both the regions. This is the first study designed to analyse the prevalence of polymorphism in the P. falciparum k13-propeller domain in the Jazan region of southwest Saudi Arabia, where malaria is endemic. METHODS: One-hundred and forty P. falciparum samples were collected from Jazan region of southwest Saudi Arabia at three different times: 20 samples in 2011, 40 samples in 2016 and 80 samples in 2020 after the implementation of ACT. Plasmodium falciparum kelch13 (k13) gene DNA was extracted, amplified, sequenced, and analysed using a basic local alignment search tool (BLAST). RESULTS: This study obtained 51 non-synonymous (NS) mutations in three time groups, divided as follows: 6 single nucleotide polymorphisms (SNPs) '11.8%' in samples collected in 2011 only, 3 (5.9%) in 2011and 2016, 5 (9.8%) in 2011 and 2020, 5 (9.8%) in 2016 only, 8 (15.7%) in 2016 and 2020, 14 (27.5%) in 2020 and 10 (19.6%) in all the groups. The BLAST revealed that the 2011 isolates were genetically closer to African isolates (53.3%) than Asian ones (46.7%). Interestingly, this proportion changed completely in 2020, to become closer to Asian isolates (81.6%) than to African ones (18.4%). CONCLUSIONS: Despite the diversity of the identified mutations in the k13-propeller gene, these data did not report widespread artemisinin-resistant polymorphisms in the Jazan region where these samples were collected. Such a process would be expected to increase frequencies of mutations associated with the resistance of ACT.

Seroprevalence of dengue fever and the associated sociodemographic, clinical, and environmental factors in Makkah, Madinah, Jeddah, and Jizan, Kingdom of Saudi Arabia.

This study aimed to estimate the seroprevalence of anti-dengue IgG antibodies in Makkah, Al Madinah, Jeddah, and Jizan; and to identify the associated demographic, clinical, and environmental independent risk factors. A community-based household serosurvey conducted between September 20, 2016 and January 31, 2017. A multi-stage stratified cluster sampling was used to select 6596 participants from Makkah, Madinah, Jeddah, and Jizan. Blood samples were drawn from all participants to detect anti-dengue IgG antibodies. A semi-structured questionnaire was used to collect information on demographic, clinical, and environmental data. Multivariate logistic regression was carried out to identify independent risk factors of dengue seropositivity. The dengue seroprevalence (95% confidence intervalI) was 26.7% (25.6%, 27.8%), with the highest (33.6%) and lowest (14.8%) rates in Jizan and Madinah, respectively, and reaching 50% or more in several districts of the four cities. Demographic predictors of seroprevalence included: dwelling in Makkah (odds ratio [OR] = 2.19, p < 0.001) or Jizan (OR = 2.17, p < 0.001); older age (OR = 3.91, p < 0.001 for age>30 years); housing type (OR = 1.84 and 1.82, p < 0.001 for popular and social houses, respectively); and number of household occupants (OR = 0.86 and 0.71 for 6-10 [p = 0.042] and 11-20 [p = 0.002] occupants, respectively). Environmental predictors included the absence of pest control works in residency area (OR = 1.39, p = 0.002), presence of mosquitoes in the home (OR = 1.39, p = 0.001), and absence of awareness campaigns (OR = 1.97, p < 0.001). One in four inhabitants of the Western region of Saudi Arabia was seropositive for the dengue virus. Implementation of behavior-based educational programs is recommended, involving the population in the identification and eradication of vector sources and promoting appropriate behaviors that prevent the spread.

Perceived Barriers to Breast Cancer Screening among Saudi Women at Primary Care Setting

Background: Screening for breast cancer (BC) is of low rate in Saudi Arabia; although it is provided in the country free of charge to the population. This cross-sectional study aimed at investigating the perceived barriers towards BC screening in Al Hassa, Saudi Arabia. It is crucial for increasing the rate of utilization of screening to identify the possible barriers for seeking BC screening in order to enhance early diagnosis and improve outcome. Materials and Methods: A total of 816 adult Saudi women aged ≥ 30 years attending for routine primary health services or accompanying patients at the selected primary health care centers (PHCs) were randomly selected from 12 PHCs (8 urban and four rural) using multi-stage sampling method. Participants were invited to personal interview using semi-structured data collection instrument including inquiries about socio-demographics, reproductive history, previous histories of diagnosed breast lesions and breast cancer. The perceived individual barriers towards screening, their attitudes, the reasons for not attending previously held screening campaigns in Al Hassa, were also included. Results: Low utilization of BC screening has being significantly associated with woman's age (OR=2.55; 95% CI= 1.71-3.83), higher educational status (OR=2.98; 95% CI=2.05-4.34), higher family income (OR=1.96; 95% CI=1.31-2.93), using hormonal contraception (OR=1.46; 95% CI=0.99-2.13) and positive history of previous breast (OR=12.16; 95% CI=6.89-21.46), as shown by the results of the logistic regression model. Exploratory factor analysis showed that personal fears (especially fear of doctors/ examiners, fear of hospitals and health facilities and fear of consequences/results) were the major factors that hinder women from utilizing the free of charge BC screening with high loading eigenvalue of 3.335, explaining 30.4% of the barriers. Conclusion: Educational interventions aim at improving breast cancer knowledge and addressing barriers should be incorporated as core component of the screening program in Saudi Arabia.

HDQ, a potent inhibitor of Plasmodium falciparum proliferation, binds to the quinone reduction site of the cytochrome bc1 complex.

The mitochondrial bc(1) complex is a multisubunit enzyme that catalyzes the transfer of electrons from ubiquinol to cytochrome c coupled to the vectorial translocation of protons across the inner mitochondrial membrane. The complex contains two distinct quinone-binding sites, the quinol oxidation site of the bc(1) complex (Q(o)) and the quinone reduction site (Q(i)), located on opposite sides of the membrane within cytochrome b. Inhibitors of the Q(o) site such as atovaquone, active against the bc(1) complex of Plasmodium falciparum, have been developed and formulated as antimalarial drugs. Unfortunately, single point mutations in the Q(o) site can rapidly render atovaquone ineffective. The development of drugs that could circumvent cross-resistance with atovaquone is needed. Here, we report on the mode of action of a potent inhibitor of P. falciparum proliferation, 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ). We show that the parasite bc(1) complex--from both control and atovaquone-resistant strains--is inhibited by submicromolar concentrations of HDQ, indicating that the two drugs have different targets within the complex. The binding site of HDQ was then determined by using a yeast model. Introduction of point mutations into the Q(i) site, namely, G33A, H204Y, M221Q, and K228M, markedly decreased HDQ inhibition. In contrast, known inhibitor resistance mutations at the Q(o) site did not cause HDQ resistance. This study, using HDQ as a proof-of-principle inhibitor, indicates that the Q(i) site of the bc(1) complex is a viable target for antimalarial drug development.

Cytochrome b mutation Y268S conferring atovaquone resistance phenotype in malaria parasite results in reduced parasite bc1 catalytic turnover and protein expression.

Atovaquone is an anti-malarial drug used in combination with proguanil (e.g. Malarone(TM)) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Q(o)) site of the mitochondrial cytochrome bc(1) complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis, and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Q(o) pocket of cytochrome b, most notably near the conserved Pro(260)-Glu(261)-Trp(262)-Tyr(263) (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterization of an atovaquone-resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270-fold shift of the inhibitory constant (K(i)) for atovaquone with a concomitant reduction in the V(max) of the bc(1) complex of ∼40% and a 3-fold increase in the observed K(m) for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein content in Y268S bc(1) suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared with the 3D7 (atovaquone-sensitive) control strain in bc(1) and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes offsets the fitness cost resulting from reduced bc(1) activity.

Glycerol: an unexpected major metabolite of energy metabolism by the human malaria parasite.

BACKGROUND: Malaria is a global health emergency, and yet our understanding of the energy metabolism of the principle causative agent of this devastating disease, Plasmodium falciparum, remains rather basic. Glucose was shown to be an essential nutritional requirement nearly 100 years ago and since this original observation, much of the current knowledge of Plasmodium energy metabolism is based on early biochemical work, performed using basic analytical techniques (e.g. paper chromatography), carried out almost exclusively on avian and rodent malaria. Data derived from malaria parasite genome and transcriptome studies suggest that the energy metabolism of the parasite may be more complex than hitherto anticipated. This study was undertaken in order to further characterize the fate of glucose catabolism in the human malaria parasite, P. falciparum. METHODS: Products of glucose catabolism were determined by incubating erythrocyte-freed parasites with D-[1-13C] glucose under controlled conditions and metabolites were identified using 13C-NMR spectroscopy. RESULTS: Following a 2 h incubation of freed-P. falciparum parasites with 25 mM D-[1-13C] glucose (n = 4), the major metabolites identified included; [3-13C] lactate, [1,3-13C] glycerol, [3-13C] pyruvate, [3-13C] alanine and [3-13C] glycerol-3-phosphate. Control experiments performed with uninfected erythrocytes incubated under identical conditions did not show any metabolism of D-[1-13C] glucose to glycerol or glycerol-3-phosphate. DISCUSSION: The identification of glycerol as a major glucose metabolite confirms the view that energy metabolism in this parasite is more complex than previously proposed. It is hypothesized here that glycerol production by the malaria parasite is the result of a metabolic adaptation to growth in O2-limited (and CO2 elevated) conditions by the operation of a glycerol-3-phosphate shuttle for the re-oxidation of assimilatory NADH. Similar metabolic adaptations have been reported previously for other microaerobic/anaerobic organisms, such as yeast, rumen protozoa and human parasitic protozoa. CONCLUSION: These data highlight the need to re-evaluate the carbon and redox balance of this important human pathogen, ultimately leading to a better understanding of how the parasite is able to adapt to the variable environments encountered during parasite development and disease progression.