Current status of 4-aminoquinoline resistance markers 18 years after cessation of chloroquine use for the treatment of uncomplicated falciparum malaria in the littoral coastline region of Cameroon

ABSTRACT

The onset and rapid spread of chloroquine resistance and the introduction of amodiaquine for the treatment of uncomplicated malaria in Cameroon have influenced the proportion of Plasmodium falciparum sensitive and resistant alleles related to 4-aminoquinoline drugs. This study was undertaken to determine the prevalence of resistance markers to antimalarial 4-aminoquinolines in Douala in the Littoral Region, and Buea in the South West Region in June 2020. Dry blood spots were prepared from malaria microscopy positive cases and used for parasite DNA extraction by chelex-100 method. Plasmodium species identification was carried out by PCR amplification/agarose gel electrophoresis of 18srRNA. The Pfcrt and Pfmdr1 genes were amplified by PCR followed by restriction digestion. The prevalence of single nucleotide polymorphisms (SNPs) was compared between study sites and with previous studies carried out between 2003–2005 and 2009–2011 using the Chi square test. The results showed that Plasmodium falciparum was the dominant species occurring as mono-infections (84.6%). The wild type K76 allele of the Pfcrt gene was found in 74.9% of isolates while the wild N86, Y184 and D1246 alleles of the Pfmdr1 gene were found respectively in 87.2%, 89.6% and 100% of field isolates. The results showed a significant reduction in the mutant alleles compared to results obtained in 2003–2005 and 2009–2013. The KNYD haplotype was observed to be the most prevalent. The results indicated that there is a gradual erosion of the mutant Pfcrt and Pfmdr1 genotype and a gradual return to the sensitive P. falciparum genotype in Cameroon.

Introduction

Despite control measures aimed at reducing malaria burden, incidence continues to stagnate in the WHO African region. The region still registered an estimated 215 million malaria cases (an increase compared to the 2019 report) and about 384,000 deaths associated with malaria [1–3] [4].

Chemotherapy and vector management [4] remain the most effective tools in the management of the disease. Treatment options have rapidly evolved in Cameroon between 2002 and 2006 [5,6, 6]. Chloroquine and other 4-aminoquinolines act by accumulating in the parasite food vacuole inhibiting conversion of toxic heme to the less toxic haemozoin leading to its accumulation and subsequent death of the parasite [7]. Following a recommendation by WHO, the national malaria control program again reviewed its treatment protocol for uncomplicated falciparum malaria. It included the use of artesunate-amodiaquine (ASAQ) and artemether-Lumefantrine (AL) as the first and second line treatments respectively owing to their ability to rapidly clear fever and parasite load [5,8,9]. Although these ACTs are still very efficacious worldwide, studies carried out in South East Asia, and to a lesser extent in Africa, report emergence of parasite strains with reduced sensitivity [10] measured as delayed parasite clearance rate [10–13].

Amodiaquine, a 4-aminoquinoline, still exerts the same drug pressure as chloroquine on the parasite population [14]. It is thus important that the efficacy of amodiaquine be constantly monitored. The efficacy of amodiaquine can be monitored by assessing the key mutations associated with 4-aminoquinoline resistance. It has been established that the K76T mutation in the Pfcrt gene modulated by the N86Y, Y184F and Y1246D of the Pfmdr1 gene are associated with resistance to 4-aminoquinolines such as chloroquine and amodiaquine [15,16]. Evidence from other countries such as Malawi and Kenya, principally in the Eastern region of Africa, suggests that removal of chloroquine drug pressure from the population led to a very significant return to the sensitive genotype [17,18]. Preliminary studies by Moyeh et al [19] with field isolates collected in 2013 showed a significant reduction and gradual reemergence of molecular markers of resistance to 4-aminoquinolines in the equatorial forest region of Cameroon, compared to results obtained from East Africa [17]. The objective of this study was to assess the prevalence of key markers to 4-aminoquinolines in the present day in the Atlantic coastline regions of Cameroon, and to compare the findings with existing data derived from similar studies 18 years ago.

Methodology

This study was carried out in two major cities: Douala and Buea. The towns are in the Littoral and South West Regions of Cameroon respectively. Buea is located in Fako Division of the South West Region of Cameroon. Douala, the largest city in Cameroon, is the capital of the Littoral Region and the economic capital of Cameroon. Buea and Douala are both located in the tropical rain forest region along the Atlantic coastline of Cameroon.

Ethical approval to carry out this study was obtained from the Faculty of Health Sciences Institutional Review Board (2018/125/UB/SG/IRB/FHS) of the University of Buea, while Administrative Authorization was obtained from the South West and Littoral Regional Delegations of Public Health (B11/MINSANTE/SWR/RDPH/P5/55P/771). Sample/data was collected from consenting individuals and for minors; assent/consent was obtained from a parent or legal guardian. The consent form was clearly written in simple English or French and those who gave their consent had to sign or apply a thumbprint on the form, or had an independent person witness the process.

Demographic and clinical data were also obtained from study participants. Children less than 5 months old, pregnant women and individuals with signs and symptoms suggestive of severe malaria were excluded from the study. Participants were randomly enrolled into the study irrespective of sex and age but in accordance with the exclusion criteria above. About 1 ml blood sample was collected by venipuncture to prepare thick smears on a clean grease free pre-labeled microscope slide. The slides were stained with freshly prepared 10% Giemsa, and viewed under oil immersion in a light microscope to detect malaria parasites and determine parasite density. Malaria parasite density was determined by counting the number of parasites in 200 white blood cells assuming there are 8,000 white blood cells per microliter of blood [20]. Parasitized blood samples were later spotted on sterile pre-labeled filter paper (Whatmann® No. 3, Sigma-Aldrich, Germany), allowed to air dry within 4 hours or overnight, and preserved in a sealed envelope with a desiccant. These filter papers were later transported to the Cell and Molecular Biology Laboratory of the Biotech Unit, University of Buea, for further analysis.

Genomic DNA was extracted from filter paper stained blood samples by boiling in Chelex®-100 (Bio-Rad, Berkeley California, USA) as previously described [21]. Plasmodium species identification by nested PCR was carried out using primers as previously described by Snounou et al. [22]. Amplified fragments were separated on a 2.5% agarose gel stained with ethidium bromide and visualized using a gel documentation system (Molecular Imager® Gel Doc^TM XR+ System with Image Lab^TM Software, Bio-Rad, Berkeley California, USA). Samples with negative results were re-extracted and re-amplified. Only P. falciparum mono-infections were used for the analysis of SNPs associated with resistance to 4-aminoquinolines. Fragments of the gene encompassing the SNP of interest in the Pfcrt and Pfmdr1 genes were amplified by nested PCR using sequence specific primers obtained from Inqaba Biotech (Pretoria, South Africa). The amplified fragments of these genes were analyzed by RFLP using the enzymes Apo1, AflIII, Dra1 and Bgl11 respectively; as previously described by Djimde et al., 2001 [11]. The restricted fragments were resolved on a 2.5% ethidium bromide stained agarose gel and visualized in a gel documentation system (Molecular Imager® Gel Doc^TM XR+ System with Image Lab^TM Software, Bio-Rad, Berkeley California, USA).

Data generated in this study was entered into Microsoft Excel, and the reconciled data was exported to GraphPad Prism (La Jolla, Ca) and analyzed. Baseline demographic and clinical characteristics of the study participants were analyzed using descriptive statistics (means, standard deviations, etc.). Differences in proportions were analyzed using a Chi square test. Haplotypes were constructed by aligning the SNPs obtained for each parasite in the three different fragments of Pfmdr1 gene, grouping parasites with similar haplotypes together. In the analysis of the prevalence of resistance markers, SNPs with mixed alleles/genotypes (harboring both the wild type and mutant SNP) were considered mutant, whereas in haplotype analysis, such mixed infections were excluded from analysis. Statistical significance threshold was set at 5%.

Results

A total of 456 participants were screened in both towns for the presence of the malaria parasite by light microscopy. Samples/data was collected from only 240 participants (52.6%) that were shown to be parasite positive by microscopy. Of the 240 participants retained for the study, 58.8% (141/240) were females while 41.2% (99/240) were males. The proportion of females was significantly higher when compared to that of males (P = 0.0002). The participants’ ages ranged from 1–70 years with a mean age of 25.6 ± 18.9. Fever defined as temperature above or equal to 37.5°C was observed in 43.3% (104/240) of participants, and the temperature ranged from 36.0°C to 40.0°C [Median: 37.8°C, (25^th percentile: 37°C; 75^th percentile: 38°C)]. Asexual parasitemia detected by light microscopy ranged from 40 to 156,000 parasites/µl of blood.

A total of 84.6% (203/240) of field isolates were mono-infections with P. falciparum and 3.3% (8/240) of P. malariae. Mixed infections of P. ovale and P. falciparum (2.5% (06/240) and P. falciparum and P. malariae (9.6% (23/240) were observed (Table 1).

Table 1.

Distribution of parasite species in the different study sites.

	P. falciparum	P. malariae	P. falciparum + P. malariae	P. falciparum + P. ovale	Total samples
Buea	84.4%	5.2%	10.4%	0%	154
Douala	84.9%	0 %	8.1 %	7.0%	86
Total samples	203	08	23	06	240

Legend: The table shows percentage of each parasite species in field isolates from different sampling sites. When compared between the two study sites, the proportion of falciparum parasites was not found to be significantly different (P˃0.05).

PCR amplification of all fragments of interest within the two genes was successful in all 203 P. falciparum isolates. Table 2 shows the distribution of alleles observed for the various drug resistance markers considered.

Table 2.

Distribution of SNPs in the Pfcrt and Pfmdr1 gene in both study sites.

	K76T	N86Y	Y184F	Y1246D
Wild type	74.9%	87.8%	89.6%	100%
Mutant	13.8%	5.4%	2.5%	0.0%
Mixed	11.3%	10.8%	7.9%	0.0%

Legend: Percentages are a proportion of samples with the genotypes of interest,,of the total number of samples successfully amplified.

The wild type D1246Y allele was not found in all isolates studied. We also observed a high proportion of the sensitive genotype of both Pfcrt and Pfmdr1 genes.

Mutant parasites were considered to be those having the mutated gene as well as those with mixed genotypes. Therefore, 74.9% (152/203) of field isolates had the sensitive K76 genotype of the Pfcrt gene. The sensitive N86, Y184 and D1248 genotype of the Pfmdr1 gene were seen in 87.8% (170/203), 90% (184/203) and 100% (203/203) of field isolates respectively. When we compared the different marker genotypes in the different study sites, we observed that the proportion of parasites with the wild-type phenotype were slightly higher in isolates collected from Buea, but this difference was not significant (P > 0.05).

In haplotype analysis, we observed that the sensitive NYD genotype of the Pfmdr1 gene was found in 93.6% (162/173) of field isolates while the YYD, YFD and NFD were found in 2.9% (5/173), 2.9% (5/173) and 0.6% (1/173) of field isolates respectively. Combining haplotypes of the Pfcrt and Pfmdr1 gene, it was observed that the KNYD sensitive genotype was found in 86.7% (144/166) of field isolates while mutant TNYD, KNFD, TYYD and TYFD were found in 9% (15/166), 0.6% (1/166), 2.4% (4/166) and 1.2% (2/166) of field isolates respectively. Haplotype construction per study site also revealed a slightly higher proportion of the sensitive KNYD in Buea compared to Douala. This difference was not significant (P > 0.05).

Figure 1 shows the results of the evolution of prevalence of these mutant markers over different time periods.

Figure 1.

Evolution of pfcrt and pfmdr1 genotypes over time. Numbers on top of the bars indicate percentages of each marker genotype in the respective study period. Mbacham et al. 2010 carried out the 2005 study while the 2013 study was carried out by Moyeh et al., 2018.

Discussion

Chloroquine has been an affordable, safe and effective drug in managing uncomplicated malaria for decades in Cameroon before the onset and spread of parasite strains resistant to it. In this study, we sought to characterize key molecular markers of resistance to chloroquine in the coastal regions of Cameroon.

The results of this study showed a significant drop in the prevalence of markers of resistance to 4-aminoquinoline drugs. The K76T mutant allele was found in 97% of participants in samples collected just one year after the ban on chloroquine use [23] and by 2013, the proportion had dropped to 66.9% [24]. In the current study, the proportion has further dropped significantly to 25.1% (P < 0.00001). The rate of drop is small compared to data from Malawi Kublin et al. [25] just ten years after withdrawing chloroquine. This implies that almost 20 years down the line this reduction in prevalence in Cameroon does not seem to be at its lowest yet. This is probably because amodiaquine, which is another 4-aminoquinoline, was maintained as a partner drug to artesunate in treating uncomplicated falciparum malaria in Cameroon. In addition, in vivo results of clinical studies that were conducted in Malawi in 2006 showed that a 99% return to clinical efficacy was observed [17]. The significant drop in prevalence can be attributed in part to the removal of chloroquine pressure from the public or the high efficacy of ACTs to 4-aminoquinoline resistant parasites as well as selection of wild type alleles following treatment with artemether-lumefantrine (AL) [26–28]. These factors could have an additive effect explaining the drop. A similar trend was observed for alleles of the Pfmdr1 gene. Between 2003 and 2005, the prevalence of the mutant genotypes 86 N, 184 F and 1246Y were respectively 83.6%, 97.2% and 3.1% [23] but the figures dropped to 44.2%, 47.0% and 0.0% in samples collected between 2009 and 2013 [19]. In this study, carried out 8 years later, the results show that the 86Y, the 184 F and the 1246D mutant alleles were respectively 16.2%, 10.4% and 0.0%. This difference was observed to be highly significant (P≪0.00001).

This study showed that there is hope that chloroquine will return to clinical efficacy in the near future, and could probably be reconsidered as drug of choice in the treatment protocol. The study has also shown that the efficacy of amodiaquine (judging from the prevalence of molecular markers of resistance) as a partner drug remains very high, endorsing its choice as a partner drug of artemisinin in the treatment of uncomplicated falciparum malaria in Cameroon. The results clearly demonstrated a gradual return to the 4-aminoquinoline sensitive genotype of circulating P. falciparum populations within the study area. It showed a significant return to the wild type allele of both Pfcrt and Pfmdr1. The return to the wild type allele is similar to results obtained by Ndam et al [29] in South Eastern Cameroon as well as in East African countries such as Kenya [19] and Malawi [26]. These results are equally consistent with the findings obtained in Tanzania [29] and Uganda [30]. The D1246Y genotype completely disappeared from the study sites from 2013. Haplotype analysis showed a significant increase in the KNYD sensitive haplotype when compared to the other mutant haplotypes.

Of the malaria positive individuals who participated in the study, P. falciparum, either alone or in combination with ovale or malariae, still remains the most abundant parasite species – this is similar to the results obtained by Bigoga et al [31] (83.5% prevalence in Tiko) and Moyeh et al [31] in 2013 (82.7% prevalence in Mutengene). Malaria transmission thus appears to remain heterogeneous. An inverse relationship was seen in the different study sites where P. malariae infections were common in Buea and less common in Douala, whereas P. ovale infections were only seen in Douala. Although mono-infection with P. malariae causes mild disease that is never life-threatening, co-infection with falciparum greatly changes the manifestation dynamics through nonspecific and cross-specific responses [32] that can lead to a nephrotic syndrome. Once established, this syndrome does not respond to treatment and carries a high rate of mortality [33]. The presence of Plasmodium ovale in Douala suggests that current diagnostic and treatment protocol should in the near future consider including G6PD testing and primaquine radical cure to eliminate hypnozoites common with vivax and ovale infections. Contrary to results presented by Cho-Fru et al [34], no case of vivax malaria was seen in both study sites. Again, this result also confirms the findings by Moyeh et al [35] using a similar analysis, which was unable to obtain cases of P. vivax.

Conclusion

The results from this study showed that the combined withdrawal of chloroquine which leads to a reduction of chloroquine selection pressure and the introduction of more efficacious artemisinin-based combinations led to gradual erosion of the 4-aminoquinoline resistant strains of P. falciparum from the study sites. This gradual erosion of the resistant strains indicates a possible future return of chloroquine clinical efficacy as well as its use in routine antimalarial combinations in Cameroon.

Funding Statement

This research was supported partially through the Malaria Research Capacity Development in West and Central Africa (MARCAD) Consortium. The MARCAD consortium is funded through a grant from the Developing Excellence in Leadership, Training and Science (DELTAS) Africa Initiative (grant # DEL-15–010) to the University of Yaoundé I. The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)’s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust (grant # Wellcome Trust (DELTAS Africa) 107741/A/15/Z) and the United Kingdom (UK) government. The views expressed in this publication are those of the authors and not necessarily those of AAS, NEPAD Agency, Wellcome Trust or the UK government.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Institutional review board statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Review Committee of the Faculty of Health Science, University of Buea.

Informed consent statement

Prior to enrolment, written informed consent was obtained from all participants involved in this study. This included potential publication of study data.

Data Availability Statement

Data are contained within the article.