This study was carried out to investigate the malaria prevalence and chloroquine-resistant Plasmodium falciparum among malaria patients in Zaria. Six hundred and seventy-eight (678) patients demographic and socio-economic information was obtained using a structured questionnaire. 5% Giemsa stained thick blood films from the patients were examined using light microscopy and the samples were also confirmed for positivity using Rapid Diagnostic Test cassettes. Sucrose-sorbitol density gradient centrifugation was carried out to obtain asexual stages of the parasite. The eluent were inoculated into a complete malaria culture medium to increase the parasite density/ml. Isolation of genomic DNA extraction of Plasmodium falciparum was carried out from the culture media using the standard phenol/chloroform extraction method. Mutations associated with CQ-resistance at codon 76 of pfcrt were analyzed using nested PCR/RFLP. The isolates were genotyped for pfcrt-76 since the presence of a mutation at this codon indicates that the parasite carries a resistant gene. Of the 678 samples examined, 159 were Plasmodium slide positive indicating an overall prevalence of 23.45%. Of the 425 females examined, 120 were malaria positive while 81 out of the 253 males were positive indicating prevalence rates of 28.35% and 32.01% respectively. Mean percentage prevalence among pregnant and non-pregnant women was 50% and 26.22% respectively. RDT showed 36 positive results out 159 microscopic samples (22.64%). DNA extraction from the 36 positive samples yielded no results. To this end, CQ still elicits its therapeutic effect despite previous reports on the parasite resistance to the drug. Standard Microscopy, RDT and PCR/RFLP should be employed in diagnosis, treatment and drug resistance surveillance respectively to curtail the advent dilemma.


1.0       BACKGROUND 
Malaria remains one of the most devastating diseases of the developing world, killing 1-3million people and causing disease in 300-500 million people annually (WHO, 2003). Plasmodium falciparum is a blood-borne Apicomplexan parasite that affects persons living in sub-Saharan and other parts of the world (South East America, Asia). This parasite is responsible for most of the complication (Black water fever, gastrointestinal disturbance, hyperpyrexia, seizures, and hypoglycemia in pregnant women) associated with human malaria.

Antimalarial drug resistance is a major public health problem, which hinders the control of malaria. Drug resistance in malaria describes the knowledge about the problem and also outlines the current thinking regarding strategies to limit the advent, spread and intensification of drug-resistant malaria, thereby affecting challenges facing malaria control today (WHO, 2001). Resistance of Plasmodium falciparum to chloroquine, the cheapest and most used drug is spreading in almost all endemic countries. Chloroquine is a 4-aminoquinoline derivative of quinine, which was synthesized around 1934, has since been the most widely used antimalarial drug (Bruce-Chwatt, 1980; WHO, 2001). Chloroquine resistant Plasmodium falciparum has been described everywhere Plasmodium falciparum malaria is transmitted except for malarious area of central America, in the limited areas of the Middle East and Central Asia (Mockenhaupt, 1995). Again, many antimalarial drugs currently in use are closely related chemically and development of resistance to one can facilitate development of resistance to others (Basco, 1991).

The detection of resistance in Plasmodium falciparum to antimalarial drugs involves 4 basic methods that are employed routinely. This includes; in-vivo test, in-vitro test, animal model studies and molecular characterization (Bloland, 2001). Molecular techniques are currently the choice methods which have better promising advantages over the other three models. This molecular technique utilizes the Polymerase Chain Reaction (PCR) Restriction enzyme and Restriction Fragment Length Polymorphism (RFLP) to indicate the presence of mutation and the DNA Fragment encoding biological resistance to antimalarial drugs.(Plowe et al., 1995). This technique has become a valuable surveillance tool for monitoring the occurrence, spread or intensification of drug resistance in chloroquine, sulfadoxine-pyrimethamine, cycloguanil, mefloquine, and atovaquone which are the only drugs with known molecular markers (WHO, 2001).

Drug resistance is the ability of a parasite strain to survive and / or multiply despite the administration and absorption of a drug in equal doses of higher than those recommended but within limits of tolerance of the subject (WHO, 1963). Antimalarial drug resistance in malaria has been classified,based on in-vivo:-

(i). RI-Initial clearance of the parasite but re-appearance within a month after onset of treatments.

(ii).        RII      -A reduction in parasitaemia after treatment but failure to clear the parasite

with a subsequent rise in parasitaemia.

(iii). RIII -Severe form of resistance i.e. parasitaemia shows no significant change with treatment. (WHO, 1963; Cheesbrough,1985)

The gold standard drug for treatment of malaria was chloroquine, a 4-aminoquinoline derivative which has been characterized for its efficacy, low toxicity and

affordability (Fidock et al., 2004). Inspite of the prevalence of chloroquine resistant Plasmodium falciparum, the drug is continuously used widely especially in sub-Saharan Africa. If new antimalarial drugs have to be deployed then it must meet the requirement of rapid efficacy, minimal toxicity and low cost since cost drives choice of drugs in most developing countries (Fidock et al., 2004).

Chloroquine resistant falciparum malaria has been reported in Ibadan, South West Nigeria (Solako et al., 1987 ; Sowunmi et al., 1998; Ajayi et al., 1998; Ademowo et al., 2002; Fehintola et al., 2002; Happi et al., 2003); Jos and Zaria, North Central Nigeria (Lege-Oguntoye et al,, 1989), Calabar, Eastern Nigeria (Ezedinachi et al., 1992) and Sokoto, North Western Nigeria (Abdullahi et al., 2003).

Chloroquine resistant P. falciparum was associated with specific point mutations in the P. falciparum chloroquine-resistant transporter (pfcrt) and Plasmodium falciparum multi-drug resistant ( Pfmdr- 1) genes among children aged 1-12 years in Ibadan, Nigeria. Prevalence ranges of 53% and 40% with T76 allele of pfcrt and the Y86 allele of Pfmdr-1 respectively, have been reported (Happi et al., 2003). The acquisition of resistance by the malaria parasite is an evolutionary selection pressure and it is a stable trait in the parasite once it is acquired (Foote et al., 1990; Wellems et al., 1990).

A similar study on chloroquine resistant P. falciparum in Ilorin, Nigeria reveals that twenty-four patients (20%) showed RII response while 6 patients (5%) showed RIII response out of the 120 patients sampled with ages ranging from 6 months to 34 years. Hence, the finding suggests that there is a need for extra vigilance of Chloroquine-Resistant P. falciparum (CRPF) malaria in general and malnourished children in particular
if potentially serious complications are to be averted (Olanrewaju et al., 2001). In quantifying the economic burden of malaria in Nigeria using the willingness to pay approach, it shows that the disease imposes great burden on the society and it has an adverse effect on the physical, mental and social well being of the people and on the economic development of the nation due to poverty (Ayodele et al., 2007).


Chloroquine is one of the drugs of choice for the treatment of Malaria in Nigeria and still remains the most widely used antimalarial drug in the West African sub-region (Bruce-Chwatt, 1980; Elebiyo, 2002; F.M.H, 2009) but Chloroquine resistance has been reported in many countries in sub-saharan Africa. Thus, there is the need to update and address the situation using the newly developed and validated molecular tools used for surveillance to provide valuable information.

1.2       HYPOTHESES

i. There is no chloroquine-resistance in Plasmodium falciparum in the study area.

ii. There is no association between chloroquine-resistant Plasmodium falciparum with age.

iii. There is no significant association between chloroquine-resistant Plasmodium falciparum with sex.

iv. There is no significant association between chloroquine-resistant Plasmodium falciparum with level of parasitaemia

v. There is no significant difference between Microscopy and Rapid Diagnostic Test

1.3       AIM

To study the prevalence of chloroquine-resistant Plasmodium falciparum in falciparum malaria patients in Zaria.

1.4       OBJECTIVES

The objectives of the study are to:-

i. Determine chloroquine-resistant Plasmodium falciparum mutation in falciparum malaria patients in Zaria.

ii. Determine association between chloroquine-resistant Plasmodium falciparum with age.

iii. Determine association between chloroquine-resistant Plasmodium falciparum with sex.

iv. Determine association between chloroquine-resistant falciparum with parasitaemia.

v. Determine comparison between Microscopy and Rapid Diagnostic Test.


This study will provide baseline data that will serve as a heuristic tool for stakeholders involved in malaria eradication to meet their objectives of the Roll Back Malaria programme.

Provide also a guide or a rationale for the sales of antimalarial drugs and treatment of malaria especially Plasmodium falciparum from epidemiological surveillance data for the update on drug policies.

Contribute relevant data to the Local Government, Federal Ministry of Health and their developmental agencies on the drug status in the region.

It would also go a long way to remind practitioners on the current status of the drug in the region in annual workshops or seminars.

i. Sensitivity and specificity are of crucial importance in any identification method, because false negative and false positive results would expose the subject or individual to unnecessary drug administration.

ii. Microscopy remains the cheapest and commonly used method for diagnosis of malaria. This relies on the microscopic examination of stained blood films. Its limitation when performed even by an expert is time consuming and sensitivity of detection limited when parasitaemia is low. (Coleman et al., 2002). Hence, improved techniques such as molecular technique, immunochromatographic test (Rapid Diagnostic Test) should be employed for detection.

iii. Resistance to P. falciparum had been reported in Zaria (Lege-Oguntoye et al., 1989; Dalti, 2002).

iv. PCR/RFLP is currently in use and validated methods to establish resistance in malaria.

v. Data from Microscopy/In-vitro, In-vivo and Animal models are not reliable.

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