Antimalarial Susceptibility and Drug Resistance Characterization Studies of Plasmodium Species Isolates From Patients in Sabon-Wuse, Niger State, Nigeria
CHAPTER ONE
Objectives Of Study
The specific objectives of the study were:-
- To determine the prevalence of malaria in Umaru Musa Yaradua Memorial Hospital (UMYMH), Sabon-Wuse, Niger State, Nigeria.
- To determine the antimalarial drugs commonly prescribed in the
- To determine the type of Plasmodium species isolates among patients attending the hospital
- To determine the susceptibility of Plasmodium species isolates to some antimalarial drugs commonly prescribed in Umaru Musa Yaradua Memorial Hospital (UMYMH), Sabon-Wuse, Niger State, Nigeria.
- To characterize the multi-drug resistance factor present among the resistant Plasmodium falciparum isolates.
CHAPTER TWO
LITERATURE REVIEW
Historical Outline
The history of malaria predates humanity, as this ancient disease evolved before humans did. Malaria, a widespread and potentially lethal infectious disease, has afflicted people for much of human history, and has affected settlement patterns (Carter et al., 2002). The prevention and treatment of the disease have been investigated in science and medicine for hundreds of years, and, since the discovery of the parasite which causes it, attention has focused on its biology. These studies have continued up to the present day, since no effective Malaria vaccine has yet been developed and many of the older antimalarial drugs are losing effectiveness as the parasite evolves high levels of drug resistance. As malaria remains a major public health problem, causing 250 million cases of fever and approximately one million deaths annually, understanding its history is important.
Malaria is an ancient disease and references to what was almost certainly malaria occur in a Chinese document from about 2700 BC, clay tablets from Mesopotamia from 2000 BC, Egyptian papyri from 1570 BC and Hindu texts as far back as the sixth century BC. The early Greeks, including homer in about 850 BC, Empedocles of agrigentum in about 550 BC and Hippocrates in about 400 BC, were well aware of the characteristic poor health and enlarged spleens seen in people suffering from malaria disease living in marshy places. For over 2500 years the idea that malaria fevers were caused by miasmas rising from the swamps persisted. the word malaria comes from the Italian mal’aria meaning spoiled air, although this has been disputed. Scientific studies incriminated mosquitoes as malaria vectors, first for avian malaria by Ronald Ross in 1987 and then for human malaria by the Italian scientists Giovanni and co- workers between 1898 and 1900 (Francis, 2010).
Over a century later it seems appropriate to attribute the various discoveries concerning the malaria parasites and their transmission as follows. Laveran was the first person to find Plasmodium parasites in the blood of patients suffering from malaria in 1880. MacCullum was the first to observe and report the sexual stages of a malaria-like parasite. In 1898 Grassi, Bignami and Bastienelli were the first to demonstrate that human malaria parasites were actually transmitted by mosquitoes. Ross reported that a blood-sucking insect could not take up infective organisms from an infected individual but could also transmit them later when it fed on an uninfected host (Schalagenhauf, 2004).
The Malaria Parasites
Taxonomy
The causative agents of malaria disease are protozoa of the genus Plasmodiidae, suborder haemosporidiidae, order Coccidia. There are over 100 species of Plasmodium and these are found in the blood of mammals, reptiles and birds. They are recognized taxonomically by the presence of the two types of asexual division: schizogony, in the vertebrate host; and sporogony, in the insect vector. The great majority of malarial parasites are transmitted by mosquitoes, and the parasites of humans are exclusively transmitted by anophelines. Importantly, the parasites of humans are of two subgenera, Laverania and Plasmodium. The subgenus Plasmodium includes P. falciparum, the most pathogenic causative agent of malaria, and the closely related P. reichenowi, a parasite of the higher primates such as chimpanzee (Robert and Herbert, 2003). The subgenus Laverania includes the remaining parasites of humans, namely P. vivax, P. malariae and P. ovale. Parasites of the other mammals also fall into subgenera: Plasmodium and Vinckeia; the latter includes the parasites of lemurs and the lower mammals (Robert and Herbert, 2003).
CHAPTER THREE
MATERIALS AND METHODS
Materials
The following equipment , reagent and chemicals were used for the study.
Equipment
Tissue culture plates 12 x 8 wells (Becton Dickinsin Labware, New Jersy, model – Falcon 3072), Micropipette- 0-50 µL, 0-100 µL and 100-1000 µL (Micropet), Microscope (Fischer Scientific Company), Microscope slides, Autoclave (GULFEX Medical and Scientific, England, model – LS-B75L), Anaerobic jar (Gaspak system, model – BQBBL), Incubator (Biochemical, model SHP-250), Weighing balance (OHAUS, model – SPJ303), Sterile pipette tips and Heparinise falcum tube (Skytec Medical).
Drugs
Choroquine diphosphate powder (IPCA Laboratories Ltd), Amodiaquine dihydrochloride (Pfizer Afrique de l’Ouest) and Artesunate (Greenfield Pharm. Co. Ltd).
Chemical
RPMI 1640 liquid medium (Sigma Aldrich), Giemsa stain, Methanol (Gainland Chemical Co. United Kingdom), Buffer tablets (Hopkin and Williams, Chadwel Heath, Essex, England).
Others
Aluminium foil, Rack, plastic covered wire, Alcohol swabs, Glass writing pencil, Forceps
Methods
The Study Centre and Ethical Clearance
a. Study Centre
The study was carried out at Umaru Musa Yaradua Memorial Hospital (UMYMH), Sabon- Wuse, Niger State, Nigeria. Sabon-Wuse is a town located on Abuja-Kaduna highway with a population of 84,000 (NPC, 2007). Sabon-Wuse is the Headquarter of Tafa Local Government Area.
b. Ethical Clearance
The study protocol was approved by the Ethics Committee of Umaru Musa Yaradua Memorial Hospital Management Board (Appendix I).
CHAPTER FOUR
RESULTS
Survey of Malaria Prevalence in Sabon-Wuse
Prevalence of malaria
The total number of reported malaria cases in UMYMH, Sabon-Wuse, Nigeria was 3,350 in 2011 with an average of 275 per month. In 2012, the total number of reported cases of malaria in UMYMH, Sabon-Wuse was 3,837 with an average of 319 cases per month. Sabon- Wuse has one secondary hospital. The monthly distribution of the reported cases is presented in Fig. 4.1. The reported malaria cases in UMYMH, Sabon-Wuse increased from 3,350 cases in 2011 to 3,837 cases in 2012. That is, the rate of increase of reported malaria cases is 14.5%. The rate of increase of reported malaria cases in UMYMH, Sabon-Wuse exceeded the population growth rate reported to be between 1.93% and 2.67% in Nigeria (CIA World Factbook, 2015).
CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECOMMENDATION
Discussion
Nigeria has been reported with the largest population at risk of malaria in Africa (WHO, 2012). Malaria transmission in Nigeria takes place during both rainy and dry seasons in the southern part but have been reported to be more pronounced during rainy season in the northern region (WHO, 2012). Approximately 50% of the Nigerian population has been reported to experience at least one episode per year with an official estimate of an average of four malaria attack per person per year (WHO, 1995 and WHO, 2002).
The results of the retrospective study in this work showed that malaria patients visit hospital more during the rainy season (April to October). This rainy season avail good environmental condition for the vector of malarial parasites in Sabon-Wuse, Niger State, Nigeria.
The study of the blood samples of volunteered malarial patients showed that 54.2% of the patients that attended UMYMH had Plasmodium species in their blood. The results further showed that female (54.8%) was more infected than male (45.2%). These results point to the fact that female are more accessible to malaria parasite vector than male in the locality. The investigation further showed that 70% of sampled malarial patients were in the age group 31- 70 years with Plasmodium species malarial parasite. Plasmodium species were not detected in the stained blood smears in a high proportion of children brought to hospital with the symptom of fever. The high proportion of adult who visited UMYMH for medical advice with fever had Plasmodium species. Ramadhan et al. (2000) stated that the frequency of malaria attack depends more on the immunity of the individual and the rate of exposure to the parasites. The high incidence of P. falciparum with adult could be attributed to the high rate of exposure of this adult age group to the P. falciparum vector in Sabon-Wuse, Niger State, Nigeria.
This study also showed that the Plasmodium falciparum was 85.7% of the positive sample population, while 14.3% had mixed infection with Plasmodium malariae. The result showed that Plasmodium falciparum was the predominant Plasmodium species in Sabon-Wuse, Niger State, Nigeria.
Observation from this study also showed that 95% (19/20) of the parasites were resistant to test antimalarial drugs, while 5% (1/20) were sensitive in vitro to Chloroquine respectively. This observation is similar to Folarin et al. (2008) report that 85% (71/84) of malaria parasites in Ibadan, southwestern Nigeria were resistant to Chloroquine. Thirty-five per cent (7/20) were resistant to Amodiaquine, while 65% (13/20) of the Plasmodium falciparum parasites isolates were sensitive in vitro to Amodiaquine. This is similar to the earlier report in Southwest, Nigeria (Oyedeji et al., 2005), where resistant of Plasmodium falciparum isolate to Amodiaquine was found to be 39% (14/36). However, the result contradicts Falaki (2012) who reported that Plasmodium falciparum isolates in Kano were 100% sensitive to Amodiaquine. It was found that 25% (4/20) of the Plasmodium falciparum isolates were sensitive to Artesunate. This is similar to Na-Bangchang et al. (2013) who reported 36.7% declining in sensitivity to artesunate in Thai-Myanmar border.
Summary
The observations made in this study are as follows:-
- The total number of reported malaria cases increased from 3,350 in 2011 to 3,837 in 2012 (14.5% increase)
- Artemesinin combination therapy (ACT) are the current first line drug commonly prescribed in Umaru Musa Yardua Memorial Hospital, Sabon-Wuse, Niger State,
- Plasmodium falciparum is the predominant malaria parasite isolated from malaria patient in Sabon-Wuse.
- There is a high resistant to Chloroquine drug based on the in vitro study, which is also the least prescribed antimalarial drugs in the hospital. DNA analysis indicates the presence of Pfcrt-Ra Gene in the study area, which further confirms resistance to Chloroquine
- In vitro study shows resistant of falciparum isolate to Artesunate drug in the study area. DNA analysis indicates the presence of PfATPase Gene in the study area, which indicate resistance to Arthemisinin based drugs like Artesunate .
- Thirty-five percent of the falciparum isolates were found to be resistant to Amodiaquine drug.
- Artesunate-Amodiaquine combination shows a high sensitivity to falciparum isolates in the study area.
Conclusion
This study showed the presence of MDR, ATPase and Chloroquine resistant genes in P. falciparum isolated in Sabon-Wuse, Niger State. These genes might be responsible for resistant to some of the antimalarial drugs used in Sabon-Wuse, Niger State. Based on the findings from this study, possibility of eradicating malaria is still a problem in our environment.
Recommendation
There is the need for periodic antimalarial surveillance in order to curb the wide spread of resistance associated with antimalarial drugs in Sabon-Wuse, Nigeria.
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