Molecular Epidemiology of Malarial Parasite From Punjab, Pakistan and its Control Through Plants Based Extracts and Nanoparticles

Abstract

Malaria is one of the serious diseases distributed worldwide, especially in the underdeveloped countries of tropical and subtropical regions. It is caused by protozoan parasites of genus Plasmodium, which is transmitted in humans a mosquito vector of the genus Anopheles. In human, the five species of Plasmodium are yet diagnosed causing malaria i.e. P. vivax, P. ovale, P. malariae, P. falciparum and P. knowlesi of which P. falciparum and P. vivax are widely distributed. The available epidemiological data on malaria from Pakistan is insufficient. Moreover the malarial patients are showing resistance against the prime antimalarial drugs like chloroquine, pyrimethamine, mefloquine and artemisinin. Therefore the present work is planned to identify different species of Plasmodium, their incidence in the ten selected cities of Punjab, Pakistan situated in the Northern and Southern Punjab. The seed extract of five medicinal plants and nanoparticles of silver and iron of three effective plants that showed antiplasmodial activity were evaluated in an in vivo experiment using BALB/c mice infected with Plasmodium berghei strain. The data of (n=16075) malaria suspected cases and blood samples of microscopically positive samples (n=925) were collected from different hospitals of ten cities of Punjab. i.e. Rawalpindi, Jhelum, Gujrat, Chakwal, Gujranwala, Dera Ghazi Khan, Rajanpur, Rahim Yar Khan, Multan, and Bahawalpur in whole year of 2015 from January to December under all biosafety and bioethical rules. The blood samples were evaluated for distribution of malaria cases among the study area, seasonal variation, age group, gender, and for species diagnosis on the basis of microscopy and molecular analysis. After that prevalence was estimated on the basis of molecular results. Some of the positive PCR products were analyzed by DNA sequence and evolutionary history was inferred using the Neighbor-Joining method. The aqueous seed extracts of five medicinal plants, i.e. Trachyspermum ammi, Punica granatum, Cydonia oblonga, Benincasa hispida and Saussurea lappa were obtained via soxhelet apparatus and identified for major constituents by phytochemical analysis. The experiment was designed using BALB/c mice as a model animal infected with Plasmodium berghei pathogen to find antiplasmodial activity of selected plants. The three plants T. ammi, P. granatum and S. lappa were used to synthesize silver and iron nanoparticles. The presence of nanoparticles was confirmed by UV-visible spectroscopy; Fourier transformed infrared spectroscopy, X-ray diffraction analysis and Transmission electron microscopy. In order to calculate LD50 the mice were given four different oral doses of each plant extract, observed for their physical behavior and other toxicity symptoms like convulsion, coma and death. The LD50 was calculated by Probit analysis. The mice were grouped and infected with Plasmodium berghei. After seventy two hours of post infection mice were treated with different concentrations of experimental plants and their silver and iron nanoparticles. The effect of seed extracts and nanoparticles were studied by general symptoms, parasite count, chemosupression, survival time measurement, histopathology, biochemical and hematological analysis along with the positive and negative control on randomly selected five mice from each group. The slide positivity rate, annual parasite incidence and annual blood examination rate were 5.75%, 0.12 per 1000 population and 0.22% respectively in all recruited cities Punjab. The seasonal variation showed that highest cases were recruited in summer season, i.e. from June to September then its incidence decreases. The age wise distribution of malarial patients was maximum among 1-20 years and lowest among 41- 60 years. The gender wise distribution indicated that Plasmodium infection was dominant in males than females in all study areas. Out of the 925 microscopically positive recruited malarial infection 66.70% (n=617) were diagnosed as P. vivax, 23.67% (n=219) as P. falciparum and 9.62% (n=89) as mixed containing both P. vivax and P. falciparum whereas no case of other Plasmodium species was observed. The PCR results showed that 53.40% (n=494) as P. vivax, 16.70% (n=173) as P. falciparum and 12.86% (n=119) as mixed species and 15.02% (n=139) were not amplified through PCR. The overall prevalence of treatment seeking malarial patients in all recruited cities of Punjab was 4.88%. The DNA sequenced sample of P. vivax and P. falciparum found in this study were more related to India, Brazilian western Amazon and Korea species of phylogenetic trees. The qualitative phytochemical analysis of experimented plants revealed the presence of alkaloids, flavonoids, saponins and steroids, etc. The maximum antiplasmodial activity (80.06±4.89) was observed for the S. lappa that was treated at the dose of 750 mg/kg. The P. granatum, B. hispida, T. ammi, and C. obloga showed antiplasmodial activity as (77.58±2.09) > (71±6.12) > (69.76±2.01) > (68.76±2.01) at the dose of 500 mg/kg. The maximum antiplasmodial activity was observed for the Silver nanoparticles of P. granatum (85.92 ±0.5) > S. lappa (85.32 ±0.82) > T. ammi (83.50 ±0.65) that was treated at the dose of 150 mg/kg. The iron nano particles results showed that antiplasmodial activity was observed in an order of S. lappa (82.51±4.68), >P. granatum (81.46±3.10) > T. ammi (77.14±4.18) at the dose of 300 mg/kg. However the nonsignificant (P>0.5) results was shown when the chemosupression was compared with chloroquine treated group. The mean survival time, weight change, biochemical, the hematological and histopathological analysis also showed a marked difference in infected untreated group as compared to groups treated with nanoparticles, plant extracts and chloroquine. The slide positivity rate, annual parasite incidence, and blood examination rate was high in Northern Punjab as compared to Southern Punjab. Season wise distribution of malaria indicated the high incidence in summer and postmoon season. The incidence of malaria was high in low and middle age group as compared to the old age groups. The gender wise distribution indicated that Plasmodium infection was dominant in males. The present studies confirmed the presence of two Plasmodium species i.e. P. vivax and P. falciparum from the clinical isolates of the ten cities of Punjab, Pakistan. The overall prevalence of treatment seeking malarial patients in all recruited cities of Punjab was 4.88%. The seeds of S. lappa, P. granatum and B. hispida exhibited significantly more antiplasmodial activity as compared to T. ammi and C. oblonga. Both silver and iron nanoparticles of all the three experimented plants exhibited antiplasmodial activity and also no adverse effects on the hematology, histology and biochemical parameters were observed. It is therefore suggested that the emerging nanotechnology can be used in drug formulations. The synthesized nanoparticles can be used to develop new agents for parasite control. In this study non-specific targeting of nanoparticles were studied, but special targeted nanoparticles can be designed that can help the immune system to attack the species of malaria parasite after it enters the body and before it has a chance to hide and aggressively spread. In this way nanoparticles can also help in the vaccine formation by stimulating the immune system.