RESEARCH

 

Prevalence of Plasmodium species in asymptomatic individuals in North-Eastern South Africa: 2018 - 2019

 

 

M Munzhedzi^{I, II}; J L Guler^III; S Krivacsy^IV; P E Shifflett^V; D J Operario^VI; R Dillingham^VII; E T R McQuade^VIII; P O Bessong^{II, IX, X}

^IPhD; Department of Biochemistry and Microbiology, University of Venda, Thohoyandou, South Africa
^IIPhD; SAMRC-University of Venda Antimicrobial Resistance and Global Health Research Unit, Thohoyandou, South Africa
^IIIPhD; Department of Biology, University of Virginia, Charlottesville, Va, USA
^IVMD; Department of Public Health Sciences, University of Virginia, Charlottesville, Va, USA
^VBA; Department of Public Health Sciences, University of Virginia, Charlottesville, Va, USA
^VIPhD, MPH; Division of Infectious Diseases and International Health, University of Virginia School of Medicine, Charlottesville, Va, USA
^VIIMD, MPH; Center for Global Health Equity, Department of Infectious Diseases and International Health, University of Virginia, Charlottesville, Va, USA
^VIIIPhD; Department of Epidemiology, Emory University, Atlanta, Ga, USA
^IXPhD; Center for Global Health Equity, University of Virginia, Charlottesville, Va, USA
^XPhD; School of Health Sciences, University of KwaZulu-Natal, Durban, South Africa

Correspondence

 

 

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ABSTRACT

BACKGROUND. Asymptomatic Plasmodium infections in endemic areas pose a challenge to malaria prevention and control strategies. The Ha-Lambani area in Vhembe district, Limpopo Province, South Africa, experiences periodic malaria outbreaks, possibly influenced by asymptomatic Plasmodium infections. In addition, the identification and monitoring of the Plasmodium falciparum Kelch 13 (Pfk13) gene associated with artemisinin resistance are crucial for understanding the emergence and spread of drug-resistant malaria in endemic areas.
OBJECTIVE. To determine the prevalence of asymptomatic Plasmodium infection and Pfk13 gene polymorphisms in the Ha-Lambani area in the absence of a malaria outbreak.
METHODS. Finger-prick dried blood spots from 985 asymptomatic individuals were collected from November 2018 to May 2019. A P. falciparum-specific rapid diagnostic test (RDT) was used to test for Plasmodium infection. High-resolution melt (HRM) analysis was used to test for P. falciparum, P. ovale, P. vivax and P. malariae. The prevalence of Plasmodium infection was determined by the proportion of positive cases detected by at least one of the tests. The Pfk13 gene was amplified from P. falciparum-positive samples, sequenced by Sanger and Illumina next-generation sequencing (NGS) and analysed for genetic diversity and resistance mutations to artemisinin.
RESULTS. A prevalence of 7.1% (70/985; 95% confidence interval (CI): 0.054 - 0.087) of Plasmodium infection was observed. The dominant species was P. ovale (57.14%; n=40), followed by P. falciparum (37.1%; n=26), P. malariae (1.43%) and P. vivax (1.43%). Mixed infections were P. falciparum/P. ovale (2.9%). Plasmodium infections differed significantly by village (p<0.01). The Pfk13 gene was amplified from 5/30 (95% CI: 0.03 - 0.29). Analysis of NGS reads revealed 57 single nucleotide polymorphisms (SNPs) across the Pfk13 gene (>20% minority level). Up to 70.1% (39/57; 95% CI: 0.59 - 0.83) of the SNPs were non-synonymous and none was previously associated with artemisinin resistance. However, novel SNPs (H719Q, P701T, M472I, I526R and P443S) were detected in the propeller domain.
CONCLUSION. A relatively high asymptomatic Plasmodium infection prevalence was observed in the study area, with P. ovale being the most prevalent species. Therefore, P. ovale infections may be missed with the Plasmodium RDT. R21 and RTS,S vaccines may not offer protection against P. ovale in the study area. Further research is needed to link asymptomatic infections in the study area and the periodic malaria outbreaks, and to determine the significance of the novel SNP in the Pfk13 gene.

Keywords: asymptomatic Plasmodium infections; high-resolution melt analysis; Pfk13, NGS, malaria, Ha-Lambani, South Africa

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Malaria is a mosquito-borne disease, causing ~304 million cases and 424 700 deaths annually in sub-Saharan Africa (SSA).^[1,2] In South Africa (SA), malaria is endemic in Limpopo, KwaZulu-Natal and Mpumalanga provinces.^[3] The World Health Organization (WHO) aims to reduce the malaria incidence and mortality by 90% by 2030.^[4] Malaria symptoms range from chills, fever and headache to more severe conditions such as anaemia and brain damage.^[5-8] Plasmodium falciparum is the most common species in SA,^[5] while P. ovale, once considered rare outside West Africa, has been reported in KwaZulu-Natal and Mpumalanga.^[9,10]

Asymptomatic Plasmodium infections challenge malaria prevention efforts and SA's 2025 goal for elimination.^[11] North-Eastern SA, including the Kruger National Park, is a moderate risk area. The Ha-Lambani area borders the Park and experienced a significant increase in malaria cases from 6 to 190 between the 2015 and 2018 malaria seasons.^[12] In SA, malaria cases rise in October, peak in January and February, and decline in May.^[13] Asymptomatic infections play a role in seeding malaria outbreaks^[14] and asymptomatic parasite carriage increases transmission. Due to low parasitaemia and the absence of symptoms, detecting asymptomatic malaria is difficult, leading to missed cases.^[15,16] Limpopo harbours the highest malaria incidence rates,^[17] including a 2017 outbreak in Vhembe and Mopani (n=4 092 cases; n=33 deaths). We hypothesised that there is endemic asymptomatic Plasmodium infection in the Ha-Lambani area.

Microscopy is no longer the gold standard for malaria diagnosis;^[5,18,19] rapid diagnostic tests (RDTs)^[20] and polymerase chain reaction (PCR)-based techniques, such as high-resolution melt (HRM), are recommended. HRM offers a simple, fast workflow and high accuracy^[21] and is used to monitor asymptomatic malaria.^[22,23] Distinguishing Plasmodium species is vital for proper treatment,^[24] while identifying and monitoring the Pfk13 gene is important to understanding the emergence and spread of drug-resistant Plasmodium. This study investigated asymptomatic Plasmodium infection prevalence and the genetic diversity of the Pfk13 gene in the Ha-Lambani area of the Vhembe district of SA between 2018 and 2019.

 

Methods

The manufacturer's protocol was followed for all the kits used in this study.

Study area and consenting

A community-based study was conducted in the Ha-Lambani area (22.7108° S, 30.8442° E) from November 2018 to May 2019. The area (Fig. 1) has been described previously.^[12]

Informed consent was obtained from household heads, potential participants and legal guardians for minors. Individuals (N=985) were recruited from 261 households within Masetoni, Tshihothi and Tshamulavhu villages.

Sample collection

Finger-prick dried blood spots (DBS) were collected from 985 asymptomatic consented individuals. The procedure involved collecting four blood drops onto filter paper cards, drying them for 24 hours at room temperature and storing them in zip-top bags (with desiccants).

Rapid diagnostic test and DNA extraction

The First Response Malaria Antigen P. falciparum card test HRP2 (Premier Medical Corporation, India) (commonly used in SA health facilities) was used to screen for Plasmodium infection. Individuals who tested positive were referred for treatment. DNA was extracted from DBS using a QIAamp DNA mini kit (Qiagen, Germany).

High-resolution melt control plasmid production and purification

Plasmids of the 18S rRNA gene for P. falciparum, P. ovale, P. vivax and P. malariae (American Type Culture Collection, USA) were used as positive controls.^[21] Each species-specific plasmid was transformed into BL-21-competent bacteria using the Addgene protocol.^[25] Transformants were diluted on ampicillin-treated agar and grown (37°C) overnight. Individual colonies were isolated and expanded in Luria broth (with ampicillin), followed by plasmid purification using a QIAprep spin miniprep kit (Qiagen, Germany). The purified plasmid was stored at -20°C until used.

Polymerase chain reaction cycling, high-resolution melt and analysis

DNA samples were subjected to HRM PCR for Plasmodium speciation using the LightCycler 480 II (Roche Diagnostics, SA). The 2x HRM Type-it PCR Master mix (Qiagen, Germany) was used with primers PL1473 and PL1679^[24] in a 25 μL reaction volume. HRM, targeting the 18S rRNA, was performed as previously described.^[21] Distinct melt peaks identified species, while mixed infections were identified by multiple peaks corresponding to distinct species within a sample. HRM analysis was conducted using LightCycler 480 II software (v1.5.1), applying a 35-cycle cut-off. A participant was considered positive for Plasmodium infection if the sample was positive by at least one of the tests.

Nested polymerase chain reaction detecting the Plasmodium falciparum Kelch 13 gene

Nested PCR in a 25 μL reaction volume was used to amplify the Pfk13 gene using primers K13_Pf _F1 and K13_Pf_F2, as previously described.^[26] Amplification of Pfk13 (785 bp) was confirmed by a 2% agarose gel electrophoresis. Pfk13 amplicons were purified using the QIAquick PCR Purification Kit (Qiagen, Germany) and then subjected to Sanger and Illumina next-generation sequencing (NGS).

DNA library preparation and next-generation sequencing

Pfk13 amplicons were purified using the NucleoMag NGS Bead Suspension kit (Machery-Nagel, Germany), followed by quantification using the Qubit double-stranded high-sensitivity assay kit (Invitrogen, USA). DNA >0.2 ng/μL was normalised to an equimolar concentration of 1 M and sequenced using the Nextera XT library preparation kit (Illumina, USA). After indexing (Nextera index kit) (Illumina, USA) and clean-up, libraries were pooled to an equimolar concentration of 1 nM and sequenced on Illumina MiniSeq 500 sequencing platform (V2 kit) (Illumina, USA).

Data analysis

SPSS version 22 (IBM, USA) was used for statistical analysis. Descriptive statistics were used to describe Plasmodium infection by demographic data. A p-value of <0.05 was considered statistically significant. FastQC^[27] and Geneious (version 2023.0) (Biomatters, New Zealand) were used to assess sequence quality and analyse molecular profiles, respectively. PF3D7 1343700 K13 propeller gene sequence retrieved from the PlasmoDB was used as a reference. Minority and majority variants were called at 5% and 25%, respectively. Consensus contigs generated were aligned using Geneious multiple alignments and translated into amino acids. Phylogenetic analysis was done using maximum likelihood with 1 000 bootstraps in Geneious.

Ethical approval

Ethical approval was obtained from the University of Venda, SA (ref. no. SMNS/18/MBY/09/0507). Permission to access health centres was obtained from the Limpopo Provincial Department of Health (ref. no. LP_2018 08-020). The Ha-Lambani area leadership permitted the study in their communities.

 

Results

Demographics and rapid diagnostic test results of study participants

The study involved 985 individuals from 261 households (56.6% females; age range: 1 month - 98 years; mean age: 24.4 years; median age: 17 years; interquartile range 27). Many participants were minors aged 5 - 17 years (36.8%; 362/985). Most participants were unemployed (58.4%), had secondary school education (38.5%) and a monthly household income of <ZAR3 000 (Table 1).

Samples examined by RDT revealed 1.3% (13/985) prevalence for P. falciparum, mainly in Tshihothi and Masetoni villages (p=0.00) (Table 2).

Prevalence of Plasmodium species using molecular genotyping

HRM produced the expected distinct melting peaks at relevant melting temperatures (Fig. 2A), confirming the validity of the assay.^[21,24] This study reports a general Plasmodium prevalence of 7.1% (70/985). Most infections were with a single species (97.14%; 68/70), while in 2.86% (2/70) two Plasmodium species were detected (Fig. 2B, Table 3). P. ovale was the predominant single species detected (57.14%; 40/70), followed by P. falciparum (37.1%; 26/70), P. malariae (1.43%; 1/70) and P. vivax (1.43%; 1/70).

P. falciparum accounted for 1.3% (n=13) by HRM; three of these participant samples were detected by both RDT and HRM. Mixed infections were P. falciparum/P. ovale (2.9%; n=2).

The distribution of Plasmodium species according to participant demographics

Table 4 depicts the distribution of Plasmodium species detected by HRM, stratified by demographic factors, with more females infected with P. ovale (53.65%; 22/41) and P. falciparum (1.7%; 7/41) than males (58.6%; 17/29 and 20.6%; 6/29, respectively) (p=0.08). Infection with either P. falciparum (48.7%; 19/39) or P. ovale (53.9%; 7/13) was higher in participants aged 5 - 17 years and <5 years (p=0.38 and p=3.57), respectively.

A high prevalence of P. ovale and P. falciparum infections was observed in educated individuals (p=0.174) and higher prevalences in low-income, unemployed and grant-dependent (p=0.56) individuals. P. ovale was the most prevalent species in Ha-Lambani, with variations across villages (p=0.006).

Plasmodium prevalence at a household level

Approximately 22% (56/261; 95% CI: 0.17 - 0.27) of households had Plasmodium infection, while 85.7% (48/56) and 3.5% (2/56) had single and multiple species infection (p<0.00), respectively. Mixed infections were not statistically significant across villages (3.57%; p=0.12). However, 5.36% (3/56) of households had members infected by different Plasmodium species (p=0.00). One (0.02%) household had four P. falciparum single infections, while three P. ovale infections and one P. falciparum infection were observed in another. In Tshamulavhu and Masetoni villages there were multiple Plasmodium species infections, while there was one infection in Tshihothi village. Plasmodium infections were evenly distributed across villages, with no transmission pattern or clusters observed (Fig. 3). Notably, most households were near communal taps, which may have stagnant water over time.

Phylogenetic analysis of the Kelch 13 gene

The phylogenetic tree was determined using Pfk13 Sanger sequences from the samples of five participants. The test sequences intermingle and cluster with reference sequences from other African countries (Fig. 4).

Molecular profiles of the Plasmodium falciparum Kelch 13 gene

Five P. falciparum samples positive for the Pfk13 gene were Sanger sequenced. Only two of these (MST-080A and THT-009A) yielded high enough PCR product concentrations (>2 ng/μL) to be sequenced by NGS. Resulting sequences were used to determine Plasmodium genetic profiles. Approximately 57 SNPs were detected across the Pfk13 gene (>20% minority level, mean coverage of 60 698 and 87 257x). Of the detected SNPs, 70.1% (39/57) were non-synonymous and none was previously associated with artemisinin resistance. Several novel SNPs were detected in the Pfk13 propeller domain and 67% (26/39) of the non-synonymous SNPs (bolded in Table 5) were detected after codon 400.

 

Discussion

This study determined asymptomatic Plasmodium infection prevalence of the genetic diversity of Pfk13 in the Ha-Lambani area of SA. The prevalence of Plasmodium in the study population was 7.1% (70/985). Similarly, studies in Ethiopia and Afghanistan reported asymptomatic Plasmodium infection prevalence ranging from 6.9% to 8.2%.^[22,28,29] Contrarily, a lower Plasmodium prevalence of 2.6 - 4.2% has been observed in Ghana and Ethiopia.^[30,31] These differences may be attributed to variations in malaria endemicity, different diagnostic tests used, e.g. microscopy (less sensitive) v. RDT and PCR techniques.

Asymptomatic Plasmodium prevalence reported from KwaZulu-Natal in 2018 was 2% (~3 times lower than that observed in the current study).^[32] In Mpumalanga, however, an 8% prevalence was observed in the asymptomatic population between 2017 and 2019.^[33] KwaZulu-Natal is nearing malaria elimination, while Limpopo harbours the majority of Plasmodium infections in SA.^[11,34] The current study highlights the relatively high prevalence of asymptomatic Plasmodium infections, posing significant public health concerns and potentially hindering malaria elimination efforts in the province. Asymptomatic Plasmodium infections increase the likelihood of malaria outbreaks. The current study reported single and mixed infections. The proportion of mixed asymptomatic Plasmodium infections accounted for 2.9%, lower than the 11.8 - 24% observed in Ethiopia, KwaZulu-Natal and Nigeria.^[35-37] The rare and understudied asymptomatic P. ovale was more common, which raises concerns owing to its dormancy and relapse ability. A report from Senegal indicated 90% and 6.5% of P. falciparum and P. ovale asymptomatic infections, respectively.^[38] Also, a higher proportion of P. falciparum (92%), followed by P. malariae (4.6%), P. ovale (0.8%) and P. vivax (0.5%), was reported in Uganda.^[21] Additionally, 88.5% of malaria cases in KwaZulu-Natal were caused by P. falciparum, with P. ovale observed in mixed infections (5.9%).^[37] Interestingly, P. ovale was historically detected in SA at 9.2%^[39] in a symptomatic population of Mpumalanga. Due to its dormancy and relapse ability, P. ovale is possibly resurfacing gradually based on the relatively high prevalence observed in the current study, highlighting the need for including P. ovale antigens in common malaria RDTs. Additionally, vaccines R21 and RTS,S offer adequate protection against P. falciparum, but may not protect individuals with P. ovale infections in the current study population; hence, vaccines that include P. ovale species are recommended. Also, a follow-up study is suggested to ensure that the Plasmodium species prevalence detected was not transient. Plasmodium infection detection in the current study was significantly different by village - Tshamulavhu had more P. ovale infections than other villages (p=0.01). Notably, RDTs and HRM detected the same percentage (1.3%) of P. falciparum infections in this study.

South-East Asia's artemisinin resistance mutations are used for ongoing surveillance to detect emerging malaria parasite resistance in sub-Saharan malaria-endemic countries. Understanding Pfk13 gene mutations (associated with slow clearance of artemisinin derivatives) is crucial for tracking resistant parasites, preventing their spread and assessing control of effectiveness.^[38] Rwanda reported two mutations (R561H and P574L) on the Pfk13 gene associated with artemisinin resistance.^[40,41]

No SNP in the Pfk13 gene linked to artemisinin resistance was found in Mpumalanga.^[42] Furthermore, no Pfk13 mutations were found in Cameroon, India, Zambia, Nigeria and Botswana.^[38,43-48] However, Pfk13 mutation Q613E was detected for the first time in SA (KwaZulu-Natal) in an individual who had recently travelled to Mozambique.^[49] Moreover, the absence of known artemisinin-resistance molecular markers in Ha-Lambani isolates aligns with clinical findings, as artemisinin combination therapy (ACT) remains highly effective in Uganda, with delayed parasite clearance common.^[50] These results are encouraging and may suggest that artemisinin resistance is not yet established in Ha-Lambani. However, 57 SNPs were detected, with 39 being non-synonymous. The Pfk13 gene SNP detected after codon 400 in the Pfk13 gene is linked to delayed parasite clearance after ACT monotherapy in the greater Mekong subregion.^[42] The current study detected 23.1% of SNPs after codon 400.

This study is significant for several reasons: it examines malaria in a large group; provides seminal data on asymptomatic infection; identifies P. ovale, along with P. falciparum, as a possible major cause of malaria; highlights the limitations of current diagnostic tests; and suggests suitable techniques for malaria speciation in endemic areas. Despite these strengths, the number of Pfk13 available for analysis was small, and the finding on the genetic diversity of Pfk13 should be seen in this context. Also, it would have been of added value to characterise the full genome of P. ovale identified in the study area for the first time as a contribution to the Plasmodium genomics in SA.

 

Conclusion and recommendations

The study reveals that asymptomatic P. ovale infections may be missed by the RDT used in SA public health facilities, which target P. falciparum only. R21 and RTS,S vaccines against P. falciparum may not protect children with P. ovale infection in our study population. The relatively high level of asymptomatic Plasmodium infections indicates a potential hotspot for malaria outbreaks during transmission season. Active surveillance of Plasmodium species in asymptomatic infections is needed to predict outbreaks. The study found no Pfk13 mutations contributing to artemisinin resistance. Further research is needed to understand the significance of the identified SNPs in Pfk13 found in the study population.

Data availability. Pfk13 sequences from this study are available at the National Center for Biotechnology Information (NCBI) (BioProject ID: PRJNA1011871).

Declaration. None.

Acknowledgements. We would like to thank the residents of Ha-Lambani for contributing to the study; Ha-Lambani civic authorities for permission to conduct the study; and personnel of the Lambani Clinic for their assistance. We also thank Noah Brown of the University of Virginia for his assistance with sequence analysis. MM acknowledges predoctoral fellowship support from FIC/NIH (D43TW006578). MM benefited from a scholarship by the South African National Research Foundation (GUN108522).

Author contributions. MM, RD, ETR and POB conceived and designed the study. MM, SK and PES led field data collection. DJO, JLG and POB supervised the laboratory experiments. MM analysed the data and drafted the manuscript. POB, ETR and JLG supervised and reviewed all versions of the draft manuscript. All authors read and approved the final manuscript.

Funding. The research was supported partially by a Global Infectious Disease Research Training Program of the Fogarty International Center/ NIH (D43TW006578). Additional support was received from the University of Venda Higher Degree Committee (SMNS/18/MBY/09/0507). The funding agencies had no role in the study and publication process.

Conflicts of interest. None.

 

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Correspondence:
M Munzhedzi
holymunzhedzi@gmail.com

Received 3 October 2024
Accepted 10 March 2025