Polymorphism analysis of pfmdr1 gene in Plasmodium falciparum isolates 11 years post-adoption of artemisinin-based combination therapy in Saudi Arabia

A total of 227 Plasmodium falciparum isolates from Jazan region, southwestern Saudi Arabia were amplified for the P. falciparum multi-drug resistance 1 (pfmdr1) gene to detect point mutations 11 years after the introduction of artemisinin-based combination therapy (ACT) in Saudi Arabia. The pfmdr1 86Y mutation was found in 11.5% (26/227) of the isolates while the N86 wild allele was detected in 88.5%. Moreover, 184F point mutations dominated (86.3%) the instances of pfmdr1 polymorphism while no mutation was observed at codons 1034, 1042 and 1246. Three pfmdr1 haplotypes were identified, NFSND (74.9%), NYSND (13.7%) and YFSND (11.4%). Associations of the prevalence of 86Y mutation and YFSND haplotype with participants’ nationality, residency and parasitaemia level were found to be significant (P < 0.05). The findings revealed significant decline in the prevalence of the pfmdr1 86Y mutation in P. falciparum isolates from Jazan region over a decade after the implementation of ACT treatment. Moreover, the high prevalence of the NFSND haplotype might be indicative of the potential emergence of CQ-sensitive but artemether-lumefantrine-resistant P. falciparum strains since the adoption of ACT. Therefore, continuous monitoring of the molecular markers of antimalarial drug resistance in Jazan region is highly recommended.

The return of chloroquine-sensitive Plasmodium falciparum parasites in Jazan Region, Southwestern Saudi Arabia over a decade after the adoption of artemisinin-based combination therapy: analysis of genetic mutations in the pfcrt gene

This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11 years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%), and CVMNT single-allele (8.5%) mutant haplotypes, and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate, and nationality of the patients as well as the parasitaemia level (P < 0.05). The findings provide evidence of the potential re-emergence of CQ-susceptible P. falciparum strains in Jazan region over a decade after CQ discontinuation, with about one third of the isolates analysed carrying the pfcrt K76 CQ-sensitive wild allele and the CVMNK ancestral wild haplotype. Although the reintroduction of CQ cannot be recommended at present in Saudi Arabia, these findings support the rationale for a potential future role for CQ in malaria treatment. Therefore, continuous molecular and in-vitro monitoring mutations of pfcrt polymorphism in Jazan region is highly recommended.

Polymorphism Analysis of pfmdr1 Gene in Plasmodium falciparum Isolates 11 Years Post-adoption of Artemisinin-based Combination Therapy in Saudi Arabia

A total of 227 Plasmodium falciparum isolates from Jazan region, southwestern Saudi Arabia were amplified for the P. falciparum multi-drug resistance 1 (pfmdr1) gene to detect point mutations 11 years after the introduction of artemisinin-based combination therapy (ACT) in Saudi Arabia. The pfmdr1 86Y mutation was found in 11.5% (26/227) of the isolates while the N86 wild allele was detected in 88.5%. Moreover, 184F point mutations dominated (86.3%) the instances of pfmdr1 polymorphism while no mutation was observed at codons 1034, 1042 and 1246. Three pfmdr1 haplotypes were identified, NFSND (74.9%), NYSND (13.7%) and YFSND (11.4%). Associations of the prevalence of 86Y mutation and YFSND haplotype with participants’ nationality, residency and parasitaemia level were found to be significant (P < 0.05). The findings revealed significant decline in the prevalence of the pfmdr1 86Y mutation in P. falciparum isolates from Jazan region over a decade after the implementation of ACT treatment. Moreover, the high prevalence of the NFSND haplotype might be indicative of the potential emergence of CQ-sensitive but artemether-lumefantrine-resistant P. falciparum strains since the adoption of ACT. Therefore, continuous monitoring of the molecular markers of antimalarial drug resistance in Jazan region is highly recommended.

The return of chloroquine-sensitive Plasmodium falciparum parasites in Jazan Region, Southwestern Saudi Arabia over a decade after the adoption of artemisinin-based combination therapy: analysis of genetic mutations in the pfcrt gene

This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11 years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%), and CVMNT single-allele (8.5%) mutant haplotypes, and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate, and nationality of the patients as well as the parasitaemia level (P < 0.05). The findings provide evidence of the potential re-emergence of CQ-susceptible P. falciparum strains in Jazan region over a decade after CQ discontinuation, with about one third of the isolates analysed carrying the pfcrt K76 CQ-sensitive wild allele and the CVMNK ancestral wild haplotype. Although the reintroduction of CQ cannot be recommended at present in Saudi Arabia, these findings support the rationale for a potential future role for CQ in malaria treatment. Therefore, continuous molecular and in-vitro monitoring mutations of pfcrt polymorphism in Jazan region is highly recommended.

Antiplasmodial Activity of Ethanolic Leaf Extract of Cymbopogon citratus (DC) Stapf in Swiss Albino Mice Infected with Plasmodium berghei NK 65

The study assessed the antiplasmodial activity of the ethanolic leaf extract of Cymbopogon citratus on chloroquine sensitive Plasmodium berghei in mice. Standard methods were used to determine the bioactive components of the leaf extract, acute toxicity test and antiplasmodial activity. Mice obtained (of body weight 20-25 g) were housed and acclimatized for seven days at room temperature before the commencement of the experiment. A total of 16 albino mice were randomized into four groups of four mice each for acute toxicity while 35 were grouped into five groups of seven mice each for antiplasmodial activity. All the groups 1-5 were infected with P. berghei and were treated for six consecutive days with leaf extract dosage of 200, 400 and 800 mg/kg, standard antimalarial drug (chloroquine) as positive control and normal saline as negative control respectively. Phytochemical screening/ bioactive compounds of the leaf extract reveals the presence of saponins (10.3 mg/g), tannins (2.38 mg/g), flavonoids (1.87 mg/g), terpenoids (19.12 mg/g), steroids (6.21 mg/g) and glycosides (19.9 mg/g) as secondary metabolites. The leaf extract revealed decrease in body weight of the infected mice and did not show any toxicity at all dosage levels used. The antiplasmodial investigation revealed a decrease in percentage parasitaemia level in mice of extract treated groups compared with mice infected and not treated. The parasitaemia reduction was higher in 800 mg/kg than 200 mg/kg and 400 mg/kg. This significant decrease (P<0.05) in percentage parasitaemia level in the study was dose and time-dependent. The extract showed significant (p<0.05) antiplasmodial activity and could serve as possible candidates for the development of new effective drugs for the treatment of malaria.

Hepatozoon pyramidumi sp. n. (Apicomplexa: Adeleorina) from the blood of Echis pyramidum: morphology and SSU rDNA sequence

Hepatozoon pyramidumi sp. n. is described from the blood of the Egyptian saw-scaled viper, Echis pyramidum, captured from Saudi Arabia. Five out of ten viper specimens examined (50%) were found infected with Hepatozoon pyramidumi sp. n. with parasitaemia level ranged from 20-30%. The infection was restricted only to the erythrocytes. Two morphologically different forms of intraerythrocytic stages were observed; small and mature gamonts. The small ganomt with average size of 10.7 × 3.5 μm. Mature gamont was sausage-shaped with recurved poles measuring 16.3 × 4.2 μm in average size. Infected erythrocytes were hypertrophied; their nuclei were deformed and sometimes displaced from their central position in the normal uninfected cell. Merogonic stages were observed in the lung endothelial cell and the liver parenchyma cells. Mature meront was 17.8 × 13.6 µm and contained banana-shaped merozoites with average size of ~15 × 2 µm. Phylogenetic analysis based on the SSU rDNA sequence clustered Hepatozoon pyramidumi sp. n with previously sequenced Hepatozoon spp., most of them infected reptilian hosts without geographic consideration. The morphological and molecular comparison with closely related species proved the taxonomic uniqueness and novelty of the present form.

Molecular diagnosis of acute and chronic infection of Trypanosoma evansi in experimental male and female mice

Trypanosoma evansi is enzootic in camels in Egypt, and water buffaloes act as a reservoir for camel infection. Molecular techniques have contributed towards understanding the epidemiology of T. evansi. Trypanosoma evansi was detected in acute and chronic stages of the disease in male and female mice by polymerase chain reaction (PCR) using two primers. Two experiments were conducted. In experiment I, two groups consisting of 26 female and 26 male mice received 104 trypanosome by I/P inoculation for each mouse. In experiment II, 42 female and 42 male mice were inoculated I/P with 102 trypanosome/mouse. In addition, five mice were kept as uninfected control for each group. Mice were monitored daily for parasitaemia level during the pre-patent period using the micro-haematocrit centrifugation technique (MHCT) and conventional PCR. The primer pairs, (Trypanosoma brucei) TBR1/2 and TeRoTat1.2 (T. evansi Rode Trypanozoon antigen type [RoTat] 1.2), detected the infection after 24 hours earlier than MHCT in both experiments. The course of infection that was detected by MHCT revealed three waves of parasitaemia in female mice and two waves in male mice in the chronic stage of infection. In addition, PCR was able to detect T. evansi in different organs in the chronic stage (i.e. disappearance of parasite from blood). Application of the two primer sets on blood samples from camels showed that all samples were positive by TBR1/2 primers and only 32 of 44 were positive by TeRoTat1.2 primers. Acutely and chronically Trypanosoma-infected mice were detected by PCR in blood and organs. TBR1/2 primers were more sensitive than TeRoTat1.2 primers in detecting Trypanosoma-infected mice, and more reliable in detecting field-infected camels and excluding carrier animals.

Effect of Dietary Probiotics(Saccharomyces cerevisae)Supplementation on Serum Biochemicals ofTrypanosoma brucei bruceiInfected Rats

Background:Trypanosomosis is an important disease of both humans and animals commonly found in most parts of Africa and South America. Because of their activities, the parasites produce numerous changes in the cellular and biochemical constituents of blood. Also, trypanosomosis cause immunosuppression and also induce lipid peroxidation in the host. Probiotics confer beneficial health benefit to the host such as immune stimulation, protection against pathogens, metabolism, reduced oxidative stress, etc.Methods:Thirty (30) adult albino rats were assigned into 5 groups (A – E) of 6 rats each. Groups A, B and C rats were fed feed supplemented with probiotics at 0.08, 0.12 and 0.16 mg per kg respectively. On day 14 on the supplementation (OTS), groups A, B, C and D rats were infected with 1 x 106 trypanosomes intraperitonealy. Group E served as the not infected, not supplemented control.Results:The pre-infection supplementation did not vary the serum alanine transaminase (ALT), aspartrate transaminase (AST), urea, creatinine and total protein values of groups A, B and C. However, following infection, the ALT value of group D (infected, not supplemented) was significantly (p<0.05) higher than other groups on day 42 OTS. Also, the AST value of groups A and D were significantly (p<0.05) higher than group E but not with groups B and C on days 42 and 56 on the supplementation. On day 28 OTS, the urea level of group B was significantly (p<0.05) lower than group D whereas on days 42 and 56, group E and groups E and C were significantly (p<0.05) lower than other groups respectively. The serum creatinine level showed increase following infection with groups A and D being significantly (p<0.05) higher than other groups on days 42 and 56 OTS. On day 28 OTS, the total protein value of group A was significantly (p<0.05) lower than group C but not with other groups. By days 42 and 56 OTS, group D showed significantly (p<0.05) lower protein level when compared with other groups. The mean parasitaemia level of group D was significantly higher than other infected infected groups on days 28 and 42 on the supplementation. However, on day 56, the parasitaemia level of all infected groups did not vary (p>0.05).Conclusion:The ability of the supplementation to keep serum biochemical values before infection within range, and the subsequent maintenance of the value during most part of the infection were indication that probiotic was not toxic and may play a vital role in management of trypanosomosis.

Antiplasmodial Activity of Ethanolic Leaf Extract of Eucalyptus citriodora in Swiss Albino Mice Infected with Plasmodium berghei NK 65

Malaria is a life-threatening disease and emergence of malaria parasite resistance to antimalarial drugs, has necessitated the need for discovery and development of an alternative to malaria medicine. This study assessed the ethanolic leaf extract of Eucalyptus citriodora for the presence of bioactive components qualitatively and efficacy of the extract against the malaria parasite. Standard methods were used to determine the bioactive components of the leaf extract. Twenty (20) albino mice of body weight between 18-25 g were randomised into 5 groups of four mice each for acute toxicity test, while twenty-four (24) mice were randomised into six groups of four mice each (group 1, 2, 3, 4, 5 and 6) for antiplasmodial activity. All the groups were infected with P. berghei, except group 3 (normal control). Group 4, 5 and 6 were treated with 0.2 mL of 200, 400 and 800 mg/kg body weight of extract respectively. Group 2 (positive control) were treated with 0.2 mL of 5 mg/kg body weight of chloroquine. Group 1 (negative control) were administered with 0.2 mL of normal saline, while group 3 (normal control) were administered with 0.2 mL of normal saline for four consecutive days. Phytochemical screening revealed the presence of alkaloids, saponins, tannins, anthraquinone, flavonoids and cardiac glycosides and the extract was found safe and nontoxic. The antiplasmodial investigation revealed a decrease in percentage parasitaemia level in mice of group 4, 5 and 6 (extract treated group) compared with mice of group 1 (infected and not treated). The parasitaemia reduction was higher in group 6 (800 mg/kg). This significant decrease (P<0.05) in percentage parasitaemia level in the study was dose and time-dependent. The study revealed the potency of E. citriodora leaf extract as a future herbal candidate for the treatment of human malaria infection.