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ORIGINAL ARTICLE
Year : 2020  |  Volume : 8  |  Issue : 1  |  Page : 1-5

Phenotypic and genotypic detection of malaria parasite among patients attending Aminu Kano Teaching Hospital, Kano, Northwest Nigeria


1 Department of Medical Microbiology and Parasitology, Faculty of Clinical Sciences, College of Health Sciences, Bayero University, Kano, Kano State, Nigeria
2 Center For Dry Land Agriculture, Bayero University, Kano, Kano State, Nigeria

Date of Submission27-Oct-2019
Date of Acceptance10-May-2020
Date of Web Publication31-Jul-2020

Correspondence Address:
Mr. Abdulrazak Muhammad Idris
Department of Medical Microbiology and Parasitology, Faculty of Clinical Sciences, College of Health Sciences, Bayero University, Kano, Kano State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_28_19

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  Abstract 


Introduction: Malaria parasite (Plasmodium species) is one of the most important parasitic infections affecting people of all age groups throughout tropical countries wherever suitable hosts are found. The parasites are transmitted to man by the malaria vector, the female Anopheles mosquito during blood meals. Among all the members of the genus Plasmodium, Plasmodium falciparum is the most prevalent and common malaria species worldwide, especially in Africa and other developing countries. Aims and Objective: The present study aimed at the phenotypic and genotypic detection of malaria parasite among patients attending the General Outpatient Department of Aminu Kano Teaching Hospital, Kano, Northwest Nigeria. Material and Method: A total of 456 samples were collected from patients that voluntarily consent to participate in the study. Blood samples were randomly collected by trained personnel by vein puncture; and thick and thin smear was made on a clean glass slide and left to air dry. The smear was stained with Giemsa stain and observed under × 100 microscopic objective lens. A total of 41 (9.0%) positive malaria parasite smears were obtained with 0–9 years. Result: From 41 positive malaria parasite smears obtained by microscopic examination, 15 were randomly selected and confirmed by polymerase chain reaction (PCR) and ten were selected from negative microscopic smears. Eight (53.3%) out of the 15 positive smears and 3 (30.0%) from the negative smears were confirmed to be PCR positive. Conclusion: The finding of the study confirmed the presence of malaria parasite among the patient group in the study area using both phenotypic and genotypic detection techniques.

Keywords: Malaria, microscopy, Plasmodium, polymerase chain reaction


How to cite this article:
Adamu ZA, Idris AM, Manu AY, Umar KM. Phenotypic and genotypic detection of malaria parasite among patients attending Aminu Kano Teaching Hospital, Kano, Northwest Nigeria. Niger J Exp Clin Biosci 2020;8:1-5

How to cite this URL:
Adamu ZA, Idris AM, Manu AY, Umar KM. Phenotypic and genotypic detection of malaria parasite among patients attending Aminu Kano Teaching Hospital, Kano, Northwest Nigeria. Niger J Exp Clin Biosci [serial online] 2020 [cited 2020 Aug 8];8:1-5. Available from: http://www.njecbonline.org/text.asp?2020/8/1/1/291196




  Introduction Top


Malaria parasite (Plasmodium species) is one of the most important parasitic infections that affects people of age groups with significant impact among pregnant women and children in most of the African countries with high morbidity and mortality rates.[1] The disease is caused by the species of parasites belonging to the genus Plasmodium.[1]Plasmodium species are distributed throughout tropical countries wherever suitable hosts are found. The parasites are transmitted to man by the malaria vector, the female Anopheles mosquito during blood meals.[1] The genus Plasmodium comprises five species that can affect humans. These are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi.[1],[2]Plasmodium knowlesi is restricted to Southeast Asia. These species cause approximately 225 million infections and over 600,000 deaths/year.[1],[2] Among them, P. falciparum is the most prevalent and common malaria species worldwide, especially in Africa and other developing countries. It causes the most severe form of the disease, resulting in hundreds of thousands of deaths per year.[1],[2]

According to the World Malaria Report 2013, there were more than 200 million malaria cases in 2012.[3] An estimated 627,000 people died from malaria in 2012, with 90% of them in the sub-Saharan Africa.[3] According to the WHO,[4] Nigeria is among the endemic countries that has sufficient consistent data to evaluate trends.[4]

Kano State is located in the savanna zone of Nigeria where ecological conditions are favorable for the breeding mosquitoes. Due to the presence of numerous lakes and irrigation systems and dense population, there is an all-year-round migration and rapid urban development and increase in malaria incidence, which adversely affects the socioeconomic activities of the state.[5] The study aimed at the phenotypic and genotypic detection of malaria parasite among patients attending the General Outpatient Department (GOPD) of Aminu Kano Teaching Hospital (AKTH), Kano, Northwest Nigeria.


  Materials and Methods Top


Study area

The study was carried out in the Medical Microbiology Laboratory of AKTH located in Kano metropolis. Kano state is located in the northwest geopolitical zone of Nigeria. It comprises 44 local government areas with an estimated population of over 13 million and 20,760 km2 in area.[6] It lies between latitudes 10° 33 N to 11° 15 N and longitudes 34° CE to 8° 20 CE.[6]

Study population

This cross-sectional study consists mainly of the patients who have reported febrile illness attending the GOPD of AKTH, Kano.

Ethical clearance

Ethical permission was obtained from the ethical committee of AKTH before the commencement of the study with reference number AKTH/MAC/SUB/12A/P-3/VI/1590.

Sample collections and handling

Blood samples were collected by trained personnel through vein puncture. About 5 ml of blood samples was collected with caution from adults and children. Two drops of the blood sample were used for preparing thick and thin smear slides, and the remaining samples were preserved in an ethylenediaminetetraacetic acid container and stored at 20°C.

Laboratory investigations

Preparation of thick and thin blood smears

Both thick and thin blood films were made for this study because the thick film provides the greatest sensitivity for malaria screening, whereas the thin film provides the best detection of morphology for parasite species identification. The thick film consists of 1–2 drops of blood spread into a circle of 1.5–2.0 cm in diameter, thus allowing a large volume of blood to be examined in a single film. On comparison, the thin film is made using a single drop of blood that is spread in a layer such that the thickness decreases progressively toward the feathered edge. Both the thick and thin smears were allowed to dry completely before fixing and staining.[7]

Fixing, staining, and microscopy of blood films

The thin films were fixed in absolute methanol, and the thick films were not fixed; both the slides were left to air dry. The individual slides were placed on the staining rack, making sure that they do not touch one another. The slides were stained with 10% Giemsa solution, and the solution was gently poured on the slides to cover the smear and left for 10 min. The slides were gently rinsed with clean water and inverted in the drying rack to air dry.[8] Both the thick and thin film slides were examined at ×100 oil immersion objective lens. The thick film was examined first for the presence of parasites, and the thin film was used to identify organisms to the species level.

DNA extraction

DNA was extracted using phenol-chloroform, 200 μl of whole blood, 400 μl of lysis buffer Boston Bioproducts Ashland, United States), and 25 μl of proteinase K(Bioland Scientific LLC, Paramount, USA); mixed in the Eppendorf tube; the mixture was incubated at 65°C for 1 h; and 400 μl of phenol-chloroform (Amresco Ohio, USA) was added and vortexed for 15 s. The mixture was spun at room temperature for 10 min; the upper layer was placed into a new tube; 1000 μl of 100% ethanol (Sigma-Aldrich, St. Louis, Missouri United States) and 40 μl of 3 M sodium acetate (Amresco Ohio, USA) were added; and the tube was inverted. It was stored at −20°C overnight and then spun at 4°C for 10 min at 15,000 rpm, the upper layer was discarded, and 400μl of 70% ethanol was added. The mixture was spun at 4°C (13,000 rpm) for 5 min, and the upper layer was discarded and spun for 2 min. The remaining ethanol was removed using a pipette and allowed to dry for 15–20 min.[9]

Design of synthetic oligonucleotides (primers)

Specific primers were designed using the Primer Express Program (Applied Biosystems, Foster City, CA, USA) to hybridize the cytochrome c oxidase genes of the mitochondrial genome based on the sequence of P. falciparum, Cox III (GenBank accession No. G18346992 and M76611), as shown in [Table 1]. The amplification generated fragments of 273 and 290 bp.
Table 1: Primer sequences for Plasmodium falciparum

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Polymerase chain reaction amplification

In all cases, amplification was performed in 50 mM KCl/10 mM tris pH 8.3 (with HCl)/0.1 mg/ml gelatin/125 μM of each deoxyribonucleotide triphosphate/1.0 unit of AmpliTaq Polymerase (Perkin Elmer Cetus, USA). 17 μl of ultrapure water, 1 μl of forward primer, 1 μl of reverse primer, and 1 μl of DNA were added into a polymerase chain reaction (PCR) tube. They were well mixed by tapping the bottom of the tube and placed in a PCR machine; the machine was set and the test was run under the following conditions: 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, and then a final extension of 72°C for 4 min.[10] All amplifications were performed in PTC-100 Peltier thermal cycler (MJ Research, Cambridge, United Kingdom). The amplified products were resolved by agarose gel electrophoresis.

Gel preparation

1.5g of agarose powder was weighed and dissolved in tris-acetate buffer TAE, and the volume was made to 100 ml (in a conical flask). It was placed in the microwave for 5 min, and then removed and allowed to cool to about 45°C. 5 μl of ethidium bromide was added and shacked; it was loaded gently into the cast with the avoidance of air bubbles.

Electrophoresis

Eight microliters (8 μl) of the samples, 10 μl of the ladder marker (100 + bp), and negative control (primer + water) were loaded into the wells of the gel using a comb. The electric current was then switched on, which makes the substance in the electrophoretic field migrate toward the opposite pole based on their sizes and the charge they carry. Electrophoresis was carried out in the PowerPac 300 machine (Bio-Rad, Hercules, California, United States), immediately after electrophoresis, and the gel was stained with a fluorescent dye that binds to the nucleic acid. Exposing the stained gel to an ultraviolet light source caused the DNA to fluoresce and become visible.

Statistical analysis

Descriptive statistics of prevalence were computed with the help of Statistical package for the social sciences version 20. 0 (IBM Inc, Armonk, New York, USA).


  Results Top


Malaria prevalence was investigated among 456 patients who attended the GOPD units of AKTH. The overall prevalence of 41 (9.0%) positive malaria smears was obtained by microscopic examination. Eight (53.3%) out of 15 positives were randomly selected and 3 (30.0%) from ten negative smears were confirmed to be PCR positive [Table 2].
Table 2: Distribution of positive malaria parasite

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The age group distribution of the participants showed a high incidence of malaria among the age group of 0–9 years (28 [6.1%]) followed by the age group of 10–19 years (7 [1.5%]), with 23 (5.0%) participants. Statistically, the study showed a significant relationship between the age and incidence of the malaria parasite. The distribution of malaria parasite and gender showed a high incidence among female participants than males, with no statistically significant relationship between the two variables [Table 3].
Table 3: Demographical distribution of positive samples

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[Figure 1] and [Figure 2] show the PCR band where lane A shows the Ladder marker; lane B shows negative control lanes 1, 4, 6, 11, 13, 14, 16, 17, 18, 19; and lane 23 shows PCR-positive band annelid at 273 bp.
Figure 1: Polymerase chain reaction band

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Figure 2: Polymerase chain reaction band

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  Discussion Top


Malaria is the most prevalent tropical disease in the world today and in sub-Saharan Africa; it is ranked among the most frequent causes of morbidity and mortality, especially among pregnant women and children, and is often the leading identifiable cause.[11]

The result of this study showed that 41 (9%) of the 456 individuals examined had malaria infection; females were slightly more infected, although the difference was not statistically significant (P = 0.7518). An overall prevalence of 9.0% malaria infection was obtained in this study, which is lower than the 9.9% reported by Shehu et al.[12] in the same study area. In another study carried out by Michael et al.,[13] a high prevalence of 65.5% was recorded among patients attending the general and pediatric outpatient clinics of Federal Medical Center, Birnin Kudu, Jigawa State. Ukpai and Afoku[14] reported a prevalence of 80.3% at Imo State and 61% from Abuja.[15] Olasehinde et al.[11] also reported a prevalence rate of 80.5% among children aged <12 years in a cross-sectional study in Southwestern Nigeria. The prevalence in this study is highest in the age group of 0–9 years (6.1%). This is similar to the results of malaria prevalence in these age groups previously reported.[16],[17]

The PCR results showed that 8 (53.3%) out of the 15 positive malaria samples by microscopy that randomly selected were confirmed to be positive and 3 (30%) out of the ten that were negative by microscopy were also confirmed as PCR positive. These findings were in consonance with the study done by Hanscheid and Grobusch[18] that reported PCR detection method as a sensitive and reliable tool for detecting malaria parasite.[18]


  Conclusion Top


The present study confirmed the presence of a malaria parasite among the patient group in the study area using both phenotypic and genotypic detection techniques.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Petersen I, Eastman R, Lanzer M. Drug-resistant malaria: Molecular mechanisms and implications for public health. FEBS Lett 2011;585:1551-62.  Back to cited text no. 1
    
2.
Buppan P, Putaporntip C, Pattanawong U, Seethamchai S, Jongwutiwes S. Comparative detection of Plasmodium vivax and Plasmodium falciparum DNA in saliva and urine samples from symptomatic malaria patients in a low endemic area. Malaria J 2010;9:72.  Back to cited text no. 2
    
3.
World Health Organization. World Malaria Report 2012. WHO World Malaria Programme. Geneva, Switzerland: World Health Organization; 2013.  Back to cited text no. 3
    
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World Health Organization. World Malaria Report 2015. WHO World Malaria Programme. Geneva, Switzerland: World Health Organization; 2016.  Back to cited text no. 4
    
5.
World Health Organization. WHO Expert Committee on Malaria 20th Report. Who Technical Report Series 892. World Health Organization; 2000.  Back to cited text no. 5
    
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National Bureau of Statistics. The Latest Population Figures from National Bureau of Statistics you Need to See; Business Insider by Pulse. National Bureau of Statistics; 2018.  Back to cited text no. 6
    
7.
Mathison BA, Pritt BS. Update on malaria diagnostics and test utilization. J Clin Microbiol 2017;55:2009-17.  Back to cited text no. 7
    
8.
World Health Organization. Giemsa Staining of Malaria Blood Films. World Health Organization; 2016. p. 1-6.   Back to cited text no. 8
    
9.
Sambrook J, Frotsch EF, Maniatis T. Isolation of DNA from Mammalian Cells. Molecular Cloning. New York: Cold Spring Harbor Laboratory Press; 1989. p. 916-9.  Back to cited text no. 9
    
10.
Snounou G, Viriya S, Zhu XP, Jarra W, Rinheiro L, Do Rosario VE. High sensitivity detection of human Malaria parasite by the use of nested PCR. Mol Biochem Parasitol 1993;61:315-20.  Back to cited text no. 10
    
11.
Olasehinde GI, Ojurongbe O, Adeyeba AO, Fagade OE, Valecha N, Ayanda IO, et al.In vitro studies on the sensitivity pattern of Plasmodium falciparum to anti-malarial drugs and local herbal extracts. Mal J 2014;13:63.  Back to cited text no. 11
    
12.
Shehu A, Isah SY, Gwaram BA. Prevalence of malaria parasitaemia among blood donors in Aminu Kano teaching hospital, Kano, North-West Nigeria. Bayero J Med Lab Sci 2017;2:35-9.  Back to cited text no. 12
    
13.
Michael GC, Aliyu I, Idris U, Ibrahim H, Olalere OS, Grema BA, et al. Investigation of malaria by microscopy among febrile outpatients of a semi-rural Nigerian Medical Center: What happened to malaria control programs? Niger J General Pract 2019;17:23-30.  Back to cited text no. 13
    
14.
Ukpai OM, Afoku EL. Prevalence of malaria in Okigwe and Owerri areas of Imo state. Niger J Parasitol 2001;22:43-8.  Back to cited text no. 14
    
15.
Matur BM, Azare BA, Ugbong L. Prevalence of malaria parasite among undergraduate students of university of Abuja. Niger J Parasitol 2001;22:49-52.  Back to cited text no. 15
    
16.
Adediran, IA., Adejuyigbe, EA. and Oninla, SO. Haematological profile and Malaria parasitemia in Nigerian children requiring emergency blood transfusion. Niger J Med 2003;25:51-5.  Back to cited text no. 16
    
17.
Zaccheaus AJ, Ekanem EN. The effect of Plasmodium falciparum malaria on platelets count in patient attending the University of Calabar Teaching Hospital, Nigeria. Highland Med Res J 2005;3:9-13.  Back to cited text no. 17
    
18.
Hanscheid T, Grobusch MP. How useful is PCR in the diagnosis of malaria? Trends Parasitol 2002;18:395-8.  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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