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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 2  |  Page : 216-223

Characterization of markers of chloroquine resistance in plasmodium falciparum among pregnant women in Oyo State: Any hope of chloroquine resistance reversal?


1 Department of Medical Laboratory Science, Faculty of Basic Medical Sciences, Lead City University, Ibadan, Nigeria
2 Department of Medical Laboratory Science, School of Basic Medical Sciences, College of Medical Sciences, University of Benin, Benin City, Nigeria

Date of Submission04-Jan-2022
Date of Acceptance28-Feb-2022
Date of Web Publication17-Jun-2022

Correspondence Address:
Mufutau Mosunmade Azeez
Department of Medical Laboratory Science, Faculty of Basic Medical Sciences, Lead City University, Ibadan
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_3_22

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  Abstract 


Background: Chloroquine (CQ) which is one of the 4-aminoquinolines was once the mainstay of malaria treatment before it was officially withdrawn due to widespread resistance to it by Plasmodium species. It is one of the cheapest, safest, easily produced antimalarial compounds and has one of the longest half-lives among the antimalarial drugs which are also safe for use in pregnancy. Reversal of CQ resistance back to sensitivity has been documented after a period of withdrawal, and this may be a great relief in achievement of effective antimalarial chemotherapy at a relatively cheaper cost in Nigeria. This study investigated the characterization of markers of CQ resistance in Plasmodium falciparum infection among pregnant women in Oyo State following several years of official CQ withdrawal for treatment in Nigeria. Materials and Methods: Thick and thin blood films were made from venous blood collected from 316 consenting pregnant women and dispensed into ethylenediaminetetraacetic acid bottles after initial screening with SD Bioline RDT kit. The blood films were processed for malaria microscopy using 10% Giemsa stain. Dry blood spots on Whatman no. 1 filter paper were used for parasite DNA extraction and subsequent detection of CQ resistance markers using restriction fragment length polymorphism. Results: Eighty-two out of the 316 samples were positive for P. falciparum and subjected to molecular analysis for the detection of Pfcrt and Pfmdr1 mutant genes. Out of the 82 positive cases, 75 and 80 expressed mutant Pfcrt and Pfmdr1 genes, respectively, while 73 expressed both Pfcrt and Pfmdr1 genes. Conclusion: The high prevalence of the two major molecular markers of CQ resistance in this study, portends a concern in achieving resurgence of sensitivity after years of official withdrawal, thus official malaria management protocols should be strictly adhered to by ensuring testing before drug use while also avoiding self-medication.

Keywords: Chloroquine, molecular markers, Plasmodium falciparum, restriction fragment length polymorphism


How to cite this article:
Azeez MM, Akinbo FO. Characterization of markers of chloroquine resistance in plasmodium falciparum among pregnant women in Oyo State: Any hope of chloroquine resistance reversal?. Biomed Biotechnol Res J 2022;6:216-23

How to cite this URL:
Azeez MM, Akinbo FO. Characterization of markers of chloroquine resistance in plasmodium falciparum among pregnant women in Oyo State: Any hope of chloroquine resistance reversal?. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 Jun 26];6:216-23. Available from: https://www.bmbtrj.org/text.asp?2022/6/2/216/347715




  Introduction Top


Over the past five decades, P. falciparum which caused the most malaria incidence accounting for 99.7%,[1] has developed resistance to all antimalarial drugs used against it, be it chloroquine (CQ), sulfadoxine-pyrimethamine, quinine, mefloquine, and more recently artemisinin derivatives, thereby resulting in artemisinin combination therapy (ACT) failure.[2] It is this drug resistance that has frustrated and compromised the global onslaught against malaria through point mutations or amplification of genes.[3]

The possibility of reversal of CQ resistance to CQ sensitivity in P. falciparum is of great interest to malaria control and treatment, especially in malaria-endemic regions after the withdrawal of CQ selective pressure. Several studies have observed that Pfcrt 76T mutation can be considered the most reliable marker of CQ resistance.[4] The withdrawal of CQ for the treatment of malaria in most African countries, for example, Mozambique, led to the rapid recovery of CQ-sensitive strains of P. falciparum increasing the limit of fitness cost that accompanies CQ resistance.[5] However, this rapid reversion from CQ resistance to CQ sensitivity observed in Mozambique was not replicated in other malaria-endemic countries such as Ghana and Uganda within the same period after CQ withdrawal. This was illustrated by a study carried out in Burkina Faso where it was observed that Pfcrt 76T mutation which is the main determinant of CQ resistance decreased significantly in prevalence as compared to previous studies in the country. The same trend was observed and reported from researchers in Malawi.[4] This emerging development is very promising toward a reversal to CQ use which is cheap, safe, well tolerated, and long lasting.[6],[7]

Resistance to CQ has been shown to be primarily mediated by mutation in the Plasmodium falciparum CQ resistance transporter (Pfcrt) gene located on chromosome 7 resulting in the replacement of amino acid lysine (K) with amino acid threonine (T) at codon 76.[8],[9] Another important gene equally implicated in CQ resistance is the P. falciparum multidrug resistance gene 1 (Pfmdr1) located on chromosome 5 which codes for P-glycoprotein homolog 1[10],[11] with mutations at codons 86, 184, 1034, 1042, and 1246.[12] Out of these codon sites, codon 86 seems to have the most dominating influence since it is involved in the substrate specificity of the gene product (P-glycoprotein) and consequently alters the conveyance movement of the protein.[13],[14] Mutation at codon 86 brings about replacement of amino acid asparagine (N) with tyrosine (Y).[7] The polymorphisms in the Pfmdr1 gene were also shown to affect parasite susceptibilities to structurally diverse antimalarial compounds such as quinine, halofantrine, mefloquine, CQ, and artemisinin in vitro.[15]

The preponderance and diversity of these molecular markers of antimalarial drug resistance have made it imperative to have information, especially at site-specific or community levels, which is currently not the case in Nigeria. There is dearth of data on the prevalence and diversity of Pfcrt and Pfmdr1 mutant genes at our study sites, and hence, this study was conducted to determine the molecular markers of CQ resistance in P. falciparum among pregnant women in Oyo State, Nigeria.


  Materials and Methods Top


Study area

This study was conducted at three selected hospitals, namely: Adeoyo Maternity Hospital, Ibadan; State Hospital, Oyo; and State Hospital, Ogbomoso, in Oyo State, Nigeria. The climatic condition of this state is typically warm with a temperature range of 30°C–37°C and has two seasons, dry and rainy. The dry season is between October and April, whereas the rainy season lasts between May and September. Oyo State is an inland state in South-Western Nigeria, with its capital city at Ibadan. It is bounded in the North by Kwara State, East by Osun State, South by Ogun State, West partly by Ogun State, and the Republic of Benin. The capital of the state is Ibadan, the most populous city in black Africa. Oyo State, popularly referred to as the “Pace Setter,” is one of the 36 states of the Federal Republic of Nigeria. The state covers 28,454 km2 with an estimated population of 5,591,589.[16]

Study population

This study was conducted among pregnant women attending antenatal clinics in three selected hospitals in Oyo State, namely: Adeoyo Maternity Hospital, Ibadan (Oyo South); State Hospital, Oyo (Oyo Central); and State Hospital, Ogbomoso (Oyo North). The age of the participants ranged from 16 to 45 years. Pregnant women attending antenatal clinic and those that consented to participate were included in the study. Participants that were nonpregnant, those that refused consent, as well as antenatal patients with underlining illnesses were excluded from participation. A well-structured questionnaire bothering on biodata, sociodemographic characteristics, and antimalarial treatment/prophylaxis was administered to each participant prior to collection of specimen.

Patients consent form

The patient consent form was signed willingly by each participant after proper explanation.

Ethical approval

The protocol for this study was approved by the Ethics and Research Committee of the Ministry of Health, Ibadan, Oyo State (Reference number AD 13/479/1106 dated January 11, 2019).

Specimen collection

Approximately 4.5 ml of venous blood specimen was collected into ethylenediaminetetraacetic acid container and few drops were placed on Whatman's No. 1 filter paper, allowed to air-dry, and refrigerated in a tight-seal container containing desiccants until used for the determination of CQ-resistant genes.

Specimen processing

Initial screening

The initial screening for malaria parasite infection was carried out using Standard Diagnostic Bioline Malaria Antigen P. falciparum/Plasmodium vivax RDT test kit (05FK80) for P. falciparum and P. vivax at the three study sites' laboratories according to the manufacturer's instruction (SD Standard Diagnostics, Inc., 2015).

Microscopy

Thick and thin films were made from the blood samples on the same slide (clean grease-free frosted-end) and stained with 10% Giemsa stain as previously described.[17] The stained slides were then examined microscopically under oil immersion objective for parasite identification and speciation.

Molecular analysis (Pfcrt and Pfmdr1 mutant genes' detection)

DNA extraction, nested polymerase chain reaction (PCR), and restriction fragment length polymorphism (RFLP) were all carried out at the Biotechnology Research Laboratory of the Biochemistry Department, Federal University of Technology (FUTA), Akure, Ondo State.

DNA extraction

The trophozoite genomic DNA was extracted from the blotted drops of blood on the Whatman no. 1 filter paper using the Quick-DNA Miniprep Plus Kit from Zymo Research Corporation (Catalog Nos. D4068 and D4069) according to the manufacturer's instructions. Briefly, the blood sample spot on each filter paper was cut into small pieces into 1.0-ml Eppendorf microtube and into each tube was added: 95 μl of nuclease-free water, 95 μl of Solid Tissue Buffer, and 10 μl of proteinase K. The mixture was properly mixed on vortex mixer for 10–15 s and the microtubes were incubated at 55°C using water bath DK 420 for 3 h. The mixture was thereafter thoroughly mixed. The tubes were centrifuged using Kendro centrifuge at 12,000 x g for 1 min to remove the insoluble debris while the aqueous supernatant from each tube was transferred into a new clean microtube. Two volumes of Genomic Binding Buffer were added to the supernatant and mixed thoroughly. The mixture in each tube was transferred to a Zymo-Spin IIC-XLR Column in a collection tube and centrifuged at 12,000 ×g for 1 min. The collection tube was thereafter discarded with the flow through. To the Zymo-SpinTM II C-XLR column was added 400-μl DNA Pre-Wash Buffer in a new collection tube and centrifuged for 1 min at 12,000 ×g. The collection tube was thereafter emptied. Approximately, 700-μl gDNA wash buffer was added and centrifuged again for 1 min at 12,000 ×g. The collection tube was emptied and about 200-μl gDNA wash buffer was added and centrifuged again for 1 min. The collection tube was discarded again with the flow through. To elute the DNA, the column was transferred to a new microtube, and 50-μl DNA elution buffer was added, incubated for 5 min at room temperature, and then centrifuged at maximum speed for 1 min. The eluted DNA in each column was quantified to ascertain the purity and concentration of the DNA was extracted and then stored at −20°C until used for the PCR.

Pfcrt gene amplification and digest

The detection of mutations responsible for CQ resistance was performed by amplifying sequences marking the Pfcrt and Pfmdr1 genes using nested PCR followed by RFLP according to previously described procedures.[18] Primers used for Pfcrt K76T primary amplification included Crtp1 and Crtp2, while the secondary PCR was conducted by using the forward primer Crtp3 and the reverse primer Crtp4 [Table 1].
Table 1: Polymerase chain reaction primer sequences for amplification of codon 76 of Plasmodium falciparum chloroquine resistance transporter[19]

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Restriction fragment length polymorphism

After amplification, 20 μL of the amplicons was incubated overnight at 37°C with mutation-specific restriction enzyme Apo I. In the PCR products, the DNA sequence was cleaved at the wild-type codon site (if present) into two fragments (98 and 72 bp), while the mutant allele was not cut.

Gel electrophoresis

The digested products were separated by electrophoresis in a 2% agarose gel containing EZ-Vision, and DNA was visualized by blue-light transillumination.

Pfmdr1 gene amplification and digest

Similarly, amplification of codon 86 of the Pfmdr1 gene was conducted using nested PCR followed by restriction digest with Apol. The following primers were used: Mdr1 and Mdr2 for the primary PCR reactions and Mdr3 and Mdr4 [Table 2] for the secondary reactions after which restriction with ApoI was done. DNA fragments were compared by size and with the PCR products generated from genomic Pfmdr nested 1.
Table 2: Polymerase chain reaction primer sequences for amplification of codon 86 of Plasmodium falciparum multidrug resistance 1[18]

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Gel electrophoresis

The digested products were separated by electrophoresis in a 2% agarose gel containing EZ-Vision, and DNA was visualized by blue-light transillumination. The presence of single band is an indication of mutant Pfmdr1 among the parasitemic pregnant women.

Primers' design and synthesis

The primers were designed and synthesized by Inqaba Biotech of South Africa.

Statistical analysis

Prevalence was calculated using a simple percentage of total positives divided by total examined and multiplied by 100. All charts were created using Microsoft Excel.


  Results Top


Overall, 75 out of the 82 (91.46%) P. falciparum detected among the pregnant women in Oyo State expressed the mutant Pfcrt gene while 80 (97.56%) did same for mutant Pfmdr1 [Figure 1] and [Figure 2].
Figure 1: Frequency of mutant Pfcrt gene detection among pregnant women with Plasmodium falciparum parasitemia in Oyo State

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Figure 2: Frequency of mutant Pfmdr1 gene detection among pregnant women with Plasmodium falciparum parasitemia in Oyo State

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The expression of CQ resistance transporter (Pfcrt) gene was detected in 32 (82.05%) of the 39 pregnant women with asymptomatic P. falciparum parasitemia in Ibadan while all the 33 (100%) in Oyo and 10 (100%) in Ogbomoso expressed the gene. There was 100% expression of P. falciparum multidrug resistance 1 (Pfmdr1) gene among the Ibadan parasitemic subjects and the only positive control, while in Oyo and Ogbomoso, the expression was 96.96% and 90%, respectively. The prevalence and distribution of mutant Pfcrt and Pfmdr1 genes across the three sites and age groups are presented in [Figure 3] and [Figure 4].
Figure 3: Expression and distribution of Pfcrt and Pfmdr1 mutant genes across study sites

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Figure 4: Expression and distribution of Pfcrt and Pfmdr1 genes across age groups

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Amplification of codon 86 of the Pfmdr1 gene was carried out using nested PCR, followed by restriction digest with Apol. The presence of single band is an indication of mutant Pfmdr1 gene (86T) among the parasitemic pregnant women. The gel electrophoresis results of chloroquine resistance transporter gene (pfcrt) [Figure 5], [Figure 6] and multidrug resistance gene1 (pfmdr1) [Figure 7], [Figure 8], [Figure 9] from the three study sites as follow: the codes Y1 to Y33 represented P. falciparum obtained from Oyo township study site, while G1–G10 and B1–40 are used to denote those from Ogbomoso and Ibadan study sites, respectively. The B40 is the control with positive parasitemia. All the positive P. falciparum specimens expressed Pfmdr1 gene mutant except Y22 (from Oyo) and G5 (from Ogbomoso) that did not show visible bands meaning Pfmdr1 gene was not or poorly expressed in those parasites from those individuals.
Figure 5: Agarose gel electrophoresis results of Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) detected from Ogbomoso

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Figure 6: Agarose gel electrophoresis results of Plasmodium falciparum chloroquine resistance transporter gene detected from Ibadan

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Figure 7: Agarose gel electrophoresis results for Plasmodium falciparum multidrug-resistant 1 (Pfmdr1) gene from Oyo and Ibadan (B1-B32)

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Figure 8: Agarose gel electrophoresis results for Plasmodium falciparum multidrug-resistant 1 (Pfmdr1) gene from Ibadan (B33–B39)

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Figure 9: Agarose gel electrophoresis results for Plasmodium falciparum multidrug-resistant 1 (Pfmdr1) gene from Ogbomoso

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  Discussion Top


The interrelationship between pregnancy, P. falciparum infection, and antimalarial drug resistance serves as recipe for adverse outcomes that impact on the mother and the fetus.[20],[21] The major stumbling blocks to the total malaria control which are of severe public health concern in all malaria-endemic countries are emergence and spread of antimalarial drug resistance, and these have equally affected the epidemiology of malaria and options for its treatment.[19],[22] Determination and sustained surveillance of molecular markers of drug resistance in P. falciparum is a useful tool in monitoring malarial drug efficacy[23] and one of the proactive measures to mitigate against unpleasant consequences of malaria infection.[24]

Findings from this study demonstrate that molecular markers of resistance to CQ in P. falciparum, namely Pfcrt and Pfmdr1, are widely expressed, thereby lending credence to earlier reports.[25] The mutant gene of CQ-resistant transporter (Pfcrt) K76T associated with CQ resistance (primarily mediated by the replacement of lysine with threonine at codon 76) was expressed in 75 of the 82 (91.46%) in this study. This observation is in tandem with the result of Ojurongbe et al.[26] that reported 86% P. falciparum crt (pfcrt) mutant gene prevalence in Osogbo and 93% from Lafia. Similarly, the 95% prevalence of Pfcrt mutant gene reported by Simon-Oke et al.[15] and 94.5% by Ikegbunam et al.[27] were consistent with our finding. However, our observed prevalence is much higher than 75.9%[28] by Agomo and Oyibo and 70% by Olukosi et al reported from Lagos and 24% in Benin City metropolis respectively.[29] These reports, with that observed in this study, portend bad prognosis for early reversal to CQ sensitivity despite relative long years of official withdrawal of CQ in malaria treatment since 2005 in Nigeria against significant decrease in other climes like Cameroon after years of CQ withdrawal,[30] Malawi, just a decade after nonuse, CQ cleared 100% of asymptomatic P. falciparum infections.[31] CQ sensitivity resurgence has been envisaged based on studies elsewhere where such has been demonstrated.[5],[32] The high prevalence of mutation in the Pfcrt gene at codon 76 might be suggestive of continued drug pressure as a result of continued usage of CQ, despite official government position as was earlier suggested by Omole and Onademuren[33] and Oladipo et al.[34] The use of amodiaquine, one of the 4-aminoquinolines with structural similarity to CQ, may be another reason for the maintenance of selection pressure for resistant parasites.[19] A ray of hope for CQ sensitivity resurgence may be seen from the reported low prevalence of Pfcrt mutation (24%) in Benin City metropolis[29] which may also be replicated elsewhere. Our finding of 97.56% prevalence of Pfmdr1 mutant gene (N86Y) conflicts with 39% and 26% reported from Lafia and Osogbo, respectively, by Ojurongbe et al.,[25] 25% by Agomo and Oyibo[28] from Lagos, 18.9% from Benin,[29] 12.5% from Kaduna, 8.54% from Nnewi,[26] 4.68% from Kano, and 2% from Adamawa.[35] The high prevalence of Pfmdr1 observed in this study may foretell risk for the current ACT usage based on the suggestion that Pfmdr1 N86Y (an allele encoding tyrosine at codon 86) selection may represent a marker of tolerance to lumefantrine (one of the drugs used in ACTs). The observation in this study may be due to higher and sustained CQ usage and other structurally related antimalarial drugs (like amodiaquine) abuse which increases the mutant gene selection in the parasite. The relative cheapness, safety, and easy production protocol with long half-life are some of the qualities that endear CQ usage to the populace, especially in resource-poor and economically less endowed countries like Nigeria. Aside the aforementioned, its safety for malaria treatment caused by P. falciparum in pregnancy[36] is also an alluring factor. Preventing hypoglycemia may also stem the possibility of resistance as stimulation of gluconeogenesis occurs in response to hypoglycemia occasioned by P. falciparum infection.[37] However, efforts should also be intensified locally in phytomedicinal research for newer antimalarial drug as is being done elsewhere with some plants like Securidaca longepedunculata.[38]


  Conclusion Top


Approximately 91.5% and 97.6% Pfcrt and Pfmdr1 mutant genes, respectively, were observed in pregnant women in Oyo State. The Pfcrt 78T and Pfmdr1 86Y were observed in Ogbomoso, Oyo, and Ibadan with slightly different frequencies. The high prevalence of Pfcrt and Pfmdr1 mutant genes of CQ resistance in this study, portends a concern in achieving resurgence of sensitivity after years of official withdrawal, thus official malaria management protocols should be strictly adhered to by ensuring testing before drug use while also avoiding self-medication.

Limitation of study

This study is limited by our inability to monitor the pregnant women with positive parasitemia and expressing molecular markers of CQ resistance till delivery due to logistic reasons.

Acknowledgments

The authors owe a great debt of gratitude to the participants, the medical laboratory, and antenatal clinic staff of Adeoyo Maternity Hospital, Ibadan; State Hospital, Oyo; and State Hospital, Ogbomoso. The support and contribution of Dr. Olusola Elekofehinti of Molecular Biology Research Laboratory of Biochemistry Department of FUTA, Akure, are highly appreciated.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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