Biology and Epidemiology of Malaria Recurrence: Implication for Control and Elimination

Abstract

Malaria recurrence not only increases its clinical episodes, but also sustains transmission. It significantly contributes to a high burden of malaria and impedes elimination. Malaria recurrence can be due to reinfection, relapse, or recrudescence. Based on the type of recurrence, parasites exhibit similar or dissimilar genotypes compared to the genotype involved in initial infection. This review aimed at showing a comprehensive overview of malaria recurrence. Molecular techniques, such as real-time polymerase chain reaction (PCR), nested PCR, multiplex PCR, and sequencing, help to characterize malaria recurrence. However, these tools are hardly accessible in malaria-endemic areas and are unable to detect liver hypnozoites. Moreover, PCR is unable to adequately differentiate between relapse and reinfection of P. vivax. Recurrent malaria, particularly relapse, accounts for major portion of malaria prevalence. Through renewed parasitemia, recurrence remained as a daunting public health problem. More works remain to overcome the challenges of recurrence in efforts to control and eliminate malaria. Limited understanding of malaria recurrence impedes the development of robust tools and strategies for effective mitigation. Continued biological and epidemiological studies help unravel the persistent complexities of malaria recurrence and develop ideal tool to fight malaria.

1. Introduction

Recurrence of malaria is the return of malaria symptoms after varying lengths of symptom-free duration [1]. It often involves the reappearance of asexual stages of Plasmodium parasite in the peripheral circulation of a person, who was previously infected [1, 2, 3]. The reappearing parasite can be either similar or different to the genotype and/or species of the parasite responsible for primary infection [1, 2, 4]. Reemergence of asexual parasitemia can be due to persistence of the asexual parasite despite treatment; release from liver schizogony of reactivated hypnozoite; or from a novel infection [1].

Relapse and recrudescence are forms of recurrent malaria involving renewed parasitemia following hypnozoite reactivation and unsuccessful treatment, respectively. Reinfection involves the reappearance of malaria ensuing from the inoculation of sporozoites from an infected mosquito bite. Generally, depending on its source, malaria recurrence is classified into three types: relapse, recrudescence, and reinfection [1, 2]. The source of recurrence can be assessed by combining clinical findings with microscopy, genotyping, and measuring drug absorption [5].

2. Biology of malaria recurrence

2.1 Reinfection

Reinfection is renewed clinical illness or peripheral blood parasitemia due to a new infection with plasmodium parasite [6]. It appears after recovery from the primary infection, but usually involves different genotypes of the parasite [1, 5]. Malaria reinfection results from the injection of new sporozoites. It is critical to identify reinfection from other types of malaria recurrence for public health decision-making [5].

Markers currently employed to distinguish reinfection from other types of malaria recurrence include merozoite surface proteins (MSP1 and MSP2) and the gene of the glutamate rich protein (GLURP) [5, 7]. In reinfection, the reappearing parasite should not always exhibit a different genotype from the organism found at initial infection [1, 2]. However, all alleles of the posttreatment appearing parasite should be distinct from those identified in the pretreatment sample tested for one or more loci. This means it is possible to declare reinfection based on a single marker exhibiting disparity on all of its alleles between pretreatment and posttreatment samples [5, 7].

Reinfection is often associated with P. falciparum in endemic settings [8]. However, emerging reports show the possibility of reinfection with other species of Plasmodium [8, 9]. Likewise, Lau et al., reported reinfection with a nonhomologous strain of Plasmodium knowlesi from Malaysia [10]. In the case of Plasmodium vivax, reinfection could be disregarded if the patient relocates to settings with no malaria transmission [9]. Particular attention is vital to characterize involved species and the corresponding management [8]. Besides, reinfection further exacerbates transmission dynamics with its potential to activate hypnozoite; introduce new strains and/or species to result in heterologous relapse, and provide a chance for immune evasion. Generally, malaria recurrence increases efficiency, chance, and longevity of malaria transmission in so doing it threatens the productivity of interventions [11].

Reinfection can play a remarkable role in sustaining both the clinical and epidemiological impact of malaria. Reinfection with P. vivax entails relapse from itself and/or from the latent infection [9]. Similarly, reinfection with P. falciparum triggers relapse, thereby complicating the disease and the subsequent transmission potential [12]. In areas with high transmission of malaria, slowly eliminated antimalarial drugs are used to clear parasitemia and reduce the risk of subsequent reinfection [13]. Appropriate case management can potentially reduce parasite transmission [14]. Frequent reinfections can arise from the poor yield of interventions. Therefore, reinfection is a good marker for the effectiveness of preventive activities, such as vector control [9, 14, 15].

2.2 Relapse

Malaria relapse is hypnozoite-mediated reemergence of malaria symptoms following a successful clearance of bloodstream parasitemia. It is characterized by the recurrence of asexual parasitemia ascending from hypnozoite stages of past infection [1, 2, 3, 16]. The dormant hypnozoites persist in the liver and mature to form hepatic schizonts. These hepatic schizonts rupture and release merozoites into the peripheral circulation with an interval commonly ranging from 3 weeks to 1 year [1, 2, 17, 18]. Relapse is common in P. vivax and Plasmodium ovale infections [2, 16, 19].

Liver hypnozoites can be formed either at initial infection from the newly inoculated Plasmodium or during the existing infection from the erythrocytic Plasmodium. In areas where P. falciparum and P. vivax are co-endemic, the newly inoculated sporozoites of P. vivax sometimes directly go to the dormant stage in the liver as a strategy to avoid the potential damage that can result from the competition with P. falciparum in the bloodstream [20]. Reactivation of dormant hypnozoite occurs in response to the creation of favorable conditions due to various factors, such as a change in host immunity or inoculation of new Plasmodium [4, 12].

For a long time, relapse is considered as the hypnozoite-mediated reappearance of clinical malaria [16]. However, recent developments suggest the possibility of relapse from the reappearance of erythrocytic plasmodial stages from bone marrow and spleen. According to studies on nonhuman primates and humans, bone marrow is a key tissue reservoir of P. vivax schizonts [21, 22, 23]. Apart from a hypnozoite origin, another noncirculating source bringing about homologous parasitemia results in relapse-like P. vivax recurrences, which would better be regarded as recrudescence than relapse [6]. This concept raises the question of differentiating the parasite population arising from hypnozoite and non-hypnozoite-driven recurrences [22].

Recurrence from noncirculating and non-hypnozoite sources, adds to the longstanding complexities of malaria biology and epidemiology, particularly vivax malaria [6, 16, 19, 21, 23]. Peripheral parasitemia is a poor indicator of parasite biomass in a patient [6, 22, 23]. Therefore, assessing the involvement of the hematologic niche in malaria recurrence is gaining attention from the scientific community to improve the understanding of the pathogenesis of recurrence and reinforce the fight against malaria [3, 21, 22, 23].

Recurrences due to P. vivax can be homologous or heterologous, either of which can be reinfection, recrudescence, or relapse [4, 6, 22, 24]. Besides, different populations of hypnozoites derived from repeated inoculations can be found in the peripheral blood of a person [17]. This introduces difficulty in discerning the type of recurrence in P. vivax [4]. However, it is possible to consider most homologous recurrences as relapse provided recrudescence can be excluded in areas with high genetic diversity of P. vivax [4, 20]. Yet, relapse rates might be underrated since they can sometimes contribute to heterozygous recurrence due to polyclonal inoculum by a mosquito [4, 6, 17, 22].

The relapse of P. vivax usually ranges from weeks to a year [17, 20]. However, according to controlled human malaria infection, the frequency and number of relapses differ with host immunity and geographical location. In tropical areas, the risk of relapse is high with short intervals (less than a month); whereas temperate and subtropical areas are characterized by a lower risk of relapse recurring after a long latency (8–12 months) [17, 25]. Consistent with this, patients in regions of short relapse periodicity had a greater rate of P. vivax parasitemia than those in regions of long relapse periodicity (HR = 8.61 95% CI: 2.34–31.65; P = 0.001) [17]. In addition to places, the severity of relapse illness shortens the periodicity by activating more hypnozoites in the liver [20]. Conceivably, a modeling result shows a shorter latent period of hypnozoites increases P. vivax recurrence further reinforcing the problem (Figure 1) [26, 27].

Figure 1.

Pathogenesis of infection, and recurrence with P. vivax. Hypnozoite and schizont infection of liver occur following inoculation of sporozoites. However, after the inoculation, the parasite may develop and multiply to cause blood infection (upper row) or may enter into long-term latency (lower row). Furthermore, a portion of the hypnozoites remains in the liver to continue to relapse periodically. Suggested intervention strategies at specific weak spots are indicated (green shades inside Brocken-lined boxes). MDA: Mass drug administration.

2.3 Recrudescence

Recrudescence of malaria is the return of malaria symptoms after symptom-free periods. It is caused by parasites surviving in peripheral circulation despite treatment [1]. Recrudescence is characterized by the reappearance of genetically similar asexual stage parasites to the initial infection, usually due to incomplete therapy. In recrudescence, at least one allele at each locus is common to both paired samples collected at primary infection and recurrence [5, 16]. Recrudescence often results from incomplete clearance of asexual parasites by antimalarial drugs. It is common in infections with P. malariae and P. falciparum [1, 2].

Recrudescence associated with failure to prevent or cure malaria does not necessarily mean drug resistance. It can also be a temporary drug-associated quiescence, whereby ring stages of P. falciparum parasite in the RBCs become temporarily dormant ensuing exposure to artemisinin derivatives, such as dihydroartemisinin [28]. Based on this phenomenon, it is conceivable that early homologous recurrence of P. vivax might be misclassified as relapses while being recrudescence resulting from temporary drug-induced dormancy. In the absence of PQ therapy, the use of whole-genome analysis might help resolve such uncertainty [24]. Recrudescence should be properly characterized as it carries a considerable public health challenge (Table 1) [29].

S. No	Feature	Relapse	Reinfection	Recrudescence	References
1	Genotype similarity to primary infection	Yes/No	No	Yes	[4, 5]
2	Periodicity	Weeks to years	Depends (multifactorial)	Days to weeks; (Usually 3–4 weeks)	[9, 17]
3	Origin	Liver-hypnozoite	Sporozoites	Merozoites	[5, 6, 9]
4	Mechanism	Reactivation of liver-hypnozoite	Inoculation of sporozoites by bite from infected mosquito	Persistence of asexual parasitemia after treatment due to treatment failure	[3, 4, 5, 6]
5	Need for mosquito bite	No	Yes	No	[5, 6]
6	Commonly involved species	P. vivax and P. ovale	P. falciparum	P. falciparum and P. malariae	[5, 6]

Table 1.

Comparison of different types of recurrence in malaria.

3. Management of malaria recurrence

3.1 Diagnosis of malaria recurrence

Proper laboratory diagnosis forms the foundation for achieving malaria control and elimination. Accurate, reliable, and timely results are essential to managing initial and recurrent episodes of malaria [30, 31]. The effectiveness of malaria intervention is a function of the right diagnosis at the right time and the right treatment. Treatment without laboratory confirmation may result in overtreatment with antimalarials that may facilitate the development of drug resistance and waste resources [8, 32]. Malaria case management solely based on a clinical diagnosis not only reduces the effectiveness of treatment outcomes but also worsens the clinical and epidemiological burden of malaria by paving the way for recurrence and transmission [8].

Malaria microscopy, so far, has been the mainstay to detect and identify Plasmodium parasites. However, this method suffers from deficiency to detect very low density of parasites and difficulty to identify recurrences [33]. Molecular diagnosis provides optimum sensitivity and specificity to characterize malaria recurrence. Distinguishing recrudescence from reinfection is important to monitor the effectiveness of the therapeutic intervention. Molecular tests, such as PCR, employ genotyping target genes of the Plasmodium for diagnosis of malaria recurrence [14, 32].

Genotyping Plasmodium parasites is fundamental to comprehending parasite biology; clinical management and innovation of tools to mitigate disease. Plasmodium parasites vary by pathogenesis; susceptibility to drugs; recurrence pattern; and transmissibility by mosquito species. Genotyping these parasites help to describe the current population, understand the relationship among various populations, and characterize clinical and epidemiological features [34]. The polymerase chain reaction is a powerful tool to detect and identify Plasmodium parasites with high sensitivity and high specificity, respectively [35]. Generally, genotyping stands at the forefront of malaria elimination [5, 33, 35].

Recent advances in molecular techniques have improved the detection and identification of malaria parasites [14, 34]. Unlike microscopy and rapid diagnostic tests (RDTs), molecular techniques involving PCR help to identify and characterize recurrences [30]. They are essential partners in the control and elimination of malaria, as they both help detect and prevent recurrence through increased sensitivity and specificity [34]. They help detection of sub-microscopic, asymptomatic, mixed, and multiclonal infections in addition to suggesting drug resistance possibility [36]. Hence, molecular techniques substantially improved case management by revealing these contributors to recurrence in various ways [30, 32].

Generally, although molecular tests ominously revolutionized the interventions against malaria, some works remain to further improve their yield in the fight against malaria. There is no reliable laboratory test to diagnose liver-stage hypnozoites of P. vivax [37]. Besides, in the case of P. vivax, it is difficult to differentiate between relapse, recrudescence, and reinfection, as they might involve parasites with similar or different genotypes to the parasite found in an initial infection. Due to their high cost and technical complexity, molecular tests are not readily available in developing countries with a high burden of malaria [5, 38].

3.1.1 Nested polymerase chain reaction

Nested PCR is a type of PCR that involves two sets of primers used in two sequential runs of PCR. It is a technique, whereby the first PCR generates a mix of all Plasmodium species DNA products, which can be used in the second PCR run with primers internal (nested) to the first pair of primers. The first amplification, nest 1, allows detection of the Plasmodium genus-specific genes. Products of samples tested positive will then be subjected to a further four nested reactions (for four species) to determine the species compositions [35]. The purpose of the second run is to amplify a secondary target (species-specific sequence) within the first run product. It is an ideal technique for increasing the sensitivity and specificity of PCR [5, 33, 35].

The nPCR assay genotypes malaria parasites by targeting marker genes for 18S rRNA, merozoite surface proteins (MSP1 and MSP2), and gene of the glutamate rich protein (GLURP). Detection of amplicons from nPCR is usually done by agarose gel electrophoresis. Besides, these markers help to discriminate recrudescence from reinfection [35]. These markers possess varying power to discriminate between Plasmodium species. Accordingly, while MSP2 and GLURP have similar power, MSP1 has lower discriminatory power than these two markers. Despite the vulnerability of GLURP to “artifact bands,” and the varying performance of different markers across different geographical places, the general discriminatory power is constant [5].

Despite its remarkable benefits, nPCR is susceptible to contamination due to its extreme sensitivity and involvement in sample manipulation. Contamination originates from human errors due to the transfer of parasite material between samples or between samples and PCR reagents. The other type of contamination, which is more serious, occurs when PCR product from either of the two runs is exposed to samples, reagents, or equipment. Furthermore, the use of two separate reactions makes nPCR costly (Figure 2) [35].

Figure 2.

Schematic diagram of nested PCR. The nPCR involves two-stage amplification of the target DNA of malaria parasites by using two sets of primers that target species- and genus-level markers of the plasmodium. The external primer targets genus-level-marker: 18S rRNA, whereas the internal primer targets species-level-marker: MSP1, MSP2, and GLURP. The first run may produce unwanted products. A portion of amplicons from the first run is used as a template in each of four separate PCR reactions to produce uncontaminated final products.

3.1.2 Real-time polymerase chain reaction

Real-time polymerase chain reaction (PCR) is usually referred to as quantitative PCR (qPCR) since it involves detection, identification, and quantification of target DNA data as it occurs. It is characterized by continuous monitoring of amplicons production from the parasite DNA by using fluorescent-labeled reporters, including DNA intercalating dyes, such as SYBR green, and sequence-specific probes [39]. Real-time PCR targets polymorphic regions of MSP1, MSP2, and GLURP and are present simultaneously or singly on the Plasmodium parasite [40].

Real-time PCR can play an important role in characterizing malaria recurrence through simultaneous detection and quantification of the Plasmodium parasite. It allows multiplexing that will provide a critical framework for the identification of parasites involved in reinfection, recrudescence, and relapse. Besides, as well as can help detect genes responsible for resistance to antimalarial drugs, thereby helping the chance of malaria recurrence and transmission [41, 42, 43]. Moreover, due to its good sensitivity and specificity, real-time qPCR can be useful in both epidemiological and clinical studies as well as early detection of cases that might entail recurrence (Figure 3) [44].

Figure 3.

Graphical presentation of real-time PCR principle (TaqMan system). Real-time PCR involves a probe that is labeled at the five prime ends with a fluorescent reporter dye (F) and at the three prime ends with a quencher dye (Q). In intact probes, the fluorescence of the reporter is quenched by the close presence of a quencher. Then, probes and the complementary DNA strand are hybridized and reporter fluorescence is still quenched. During extension step of the PCR, the probe is degraded by the Taq polymerase and the fluorescent reporter is released resulting in florescence emission, which is essential information to detect and quantify the target sequence. PCR: Polymerase chain reaction, Taq pol: Taq polymerase, Q: Quencher, and F: Fluorophore [42].

3.1.3 Multiplex polymerase chain reaction

Multiplex PCR is a molecular technique characterized by simultaneous detection of several targets within a single reaction by using diverse pairs of primers specifically designed for each target [41]. In context of malaria, specie-specific primers labeled with different fluorescent dyes targeting MSP and GLURP markers are contained in the master-mix used to detect five species of Plasmodium by a single run [40]. The recently developed multiplex malaria sample ready PCR showed a hopeful result in Sierra Leone by demonstrating twice and four times higher sensitivity compared to malaria RDT and microscopy, respectively [45].

Multiplex PCR provides more information with fewer samples in a reasonably short time. It allows the detection of multiple species of Plasmodium from a single sample with a single run, thereby contributing to characterizing types of recurrence for proper case management and research purpose. In general, multiplexing is an excellent cost-saving strategy, with a particular implication in resource-limited settings [40]. However, multiplex PCR suffers from process complexity, poor universality, and variability in efficiency for different templates. Furthermore, multiplex PCR showed relatively lower sensitivity than nPCR for Plasmodium species due to the competition between different amplicons for limited supplies found in the reaction well (Figure 4) [40, 47].

Figure 4.

Diagram showing the multiplex PCR approach [46]. Multiplex PCR is a type of PCR in which multiple target DNA are simultaneously amplified in a single reaction tube. It involves the use of specific primers that can specifically combine with their corresponding DNA target contained in a master-mix, and hence allow amplification of more than one DNA fragment from a single and/or many samples.

3.1.4 Sequencing

Sequencing is the process of determining the order of nucleotides in a piece of DNA. It can be done in a small targeted genomic region or entire genome of an organism, including Plasmodium parasite [48]. A single-cell level sequencing Plasmodium parasites to explore parasite relatedness in Malawi proved the possibility of emergence of a new strain within an individual from super-infection with P. falciparum due to repeated reinfection. This study also reported unexpectedly frequent co-transmission of related parasites in intense transmission settings in the country, thereby intensifying possibility of recurrence and transmission [49].

Whole genome sequencing of P. vivax isolates from Ethiopia revealed frequent sequence and structural polymorphisms in erythrocyte binding genes that code for Duffy antigen/chemokine receptors [50]. The currently emerging data suggest that MSP involved in RBC adhesion is rapidly evolving [50, 51]. This signals for expanding pattern of P. vivax in areas previously considered not affecting Duffy-negative individuals [50]. Moreover, sequencing data can help characterize recurrence, particularly strains involved in relapse, which is inevitable since the parasite continues to infect both Duffy-negative and positive people [50, 51].

Amplicon sequencing involves sequencing a particular DNA segment of a parasite we are interested in. It helps to identify a single nucleotide polymorphism in the Plasmodium parasite [52]. Amplicon sequencing helps to characterize the genetic diversity of Plasmodium parasites by showing the number of alleles in a population in time and space. It serves as an ideal tool for characterizing the spatiotemporal flow of Plasmodium infection and geographical tracking of transmission patterns, including new and recurrence [52]. It is also an important tool to assess drug resistance genes, particularly by detecting the kelch 13 (K13) propeller gene of P. falciparum, thereby allowing recrudescence characterization [53, 54]. Furthermore, next-generation sequencing enables determining the transmission dynamics of Plasmodium species [55]. Nevertheless, these parasite sequencing methods are costly and are not sufficiently standardized for extensive use in field and clinical settings, particularly in malaria-endemic settings (Figure 5) [52, 55].

Figure 5.

Overview of DNA sequencing. DNA sequencing involves preparation of target DNA (sample collection and nucleic acid extraction); library preparation (adaptor ligation, size selection, and amplification); sequencing and data analysis (base calling, alignment, and annotation).

3.2 Treatment and/or prevention of malaria recurrence

Primaquine is the only drug widely applied to prevent relapse by clearing liver hypnozoites [27]. Recently shreds of evidence are emerging on the possibility of preventing malaria relapse with a single dose tafenoquine (TQ) treatment [56]. Universal radical cure with ACT and PQ/TQ is hoped to reduce parasitemia by leveraging the high risk of P. vivax parasitemia following P. falciparum infection in co-endemic settings [57]. This strategy produced a remarkable reward within one year by bringing about a 90% reduction in parasitemia of P. vivax among a cohort of children in Papua New Guinea [17, 58].

Preventing recurrent parasitemia reduces morbidity and mortality directly associated with malaria and indirectly with other secondary diseases [59]. Besides, a comprehensive treatment policy for malaria provides considerable benefits at an individual, public health, and operational level in settings, where P. falciparum and P. vivax are co-endemic. For prevention of recurrence, particularly relapse of P. vivax, establishing strong adherence to a full dose of PQ as a radical cure can strengthen control and elimination efforts [37]. Individuals carrying a high load of hypnozoites are more likely to relapse and therefore be targeted for treatment with PQ [60].

Improved case management is pivotal to prevention and control of malaria recurrence, particularly recrudescence. Use of highly sensitive molecular tools, such as PCR, help to detect submicroscopic parasitemia, and hence treatment [8, 31]. On the other hand, strengthening vector control strategies is important to prevent reinfection by protecting from infectious mosquito bites [8].

Hypnozoiticidal therapy against latency is used to prevent relapses from P. vivax and P. ovale [7]. The G-6-PD status of patients should guide the administration of PQ to prevent relapse. To prevent relapse, treat P. vivax or P. ovale malaria in children and adults with a 14-day course (0.25–0.5 mg/kg/BW/day) of PQ in all transmission settings. Nevertheless, this recommendation excludes pregnant women, infants aged below 6 months, women breastfeeding infants aged below 6 months, women breastfeeding older infants with unknown G-6-PD status, and people with G-6-PD deficiency. In people with G-6-PD deficiency, with close medical supervision for the risk of PQ-provoked hemolysis, consider preventing relapse by giving PQ base at 0.75 mg/kg/BW/week for 8 weeks. If the G-6-PD status is unknown and G-6-PD testing is not available, a decision to prescribe PQ must be based on risks and benefits [37, 61].

Tafenoquine is a single-dose (300 mg) drug approved only for use in combination with chloroquine (CQ) for adult patients who are nonpregnant and G-6-PD-normal. Before administering TQ , make sure the patient has normal G-6-PD activity (>70% [7, 61]. In pregnant or breastfeeding women, consider weekly chemoprophylaxis with CQ until delivery and breastfeeding is completed. Then, based on their G-6-PD status, treat with PQ to avoid future relapse [61].

4. Epidemiology of malaria recurrence

4.1 Prevalence of malaria recurrence

4.1.1 World

Malaria recurrence is a daunting public health problem worldwide. It increases the incidence and prevalence of malaria [9, 26, 60]. In many regions, a large portion of P. vivax prevalence is attributed to relapse [9, 16, 62]. A long-term study conducted on Thai-Myanmar border revealed a higher rate of P. vivax than P. falciparum recurrence, with a cumulative proportion of 31.5% (95% CI: 30.1–33.0%) and 21.5% (95% CI: 20.3–22.8%) recurrence by day 63, respectively [63].

In the recent meta-analysis by Hossain et al., recurrent parasitemia of Plasmodium species between day 7 and 42 was documented among 13.2% of patients worldwide, mainly of P. vivax recurrence. In the same report, the cumulative risks of recurrent parasitemia of any Plasmodium species by day 28, 42, and 63 were 8.1% (95% CI: 7.7–8.6), 16.8% (95% CI: 16.2–17.5), and 30.5% (95% CI: 29.4–31.6), respectively [12]. Additionally, a comprehensive review involving many studies across Asia, the Americas, and Africa showed a recurrence of P. vivax in 30% of people within 2 months after ACT [64]. In India, a country with a high burden of P. vivax region, 17.1% average relapse rates of P. vivax with quickly recurring type have been recorded from 2001 to 2003 [65]. Likewise, in Brazil, 23% of reported cases of P. vivax malaria relapse [66].

Relapse persisted as the contributor to the overall prevalence of recurrent malaria worldwide. A genotyping and whole-genome sequencing conducted in Cambodia for assessing the efficacy of CQ against P. vivax indicated that two-thirds of the recurrent P. vivax parasites resulted from heterologous relapses [67]. The incidence density of P. vivax recurrence was 45.1/100 patient-years, mostly occurring between the 4th and 13th week after initiating treatment [66]. The cumulative incidence of the first recurrence of P. vivax infection among patients receiving CQ and PQ in Turbo Municipality, Colombia, was 24.1% (95% CI: 14.6–33.7%). The majority (65.5%) of these recurrences were classified as relapse and occurred within 51 to 110 days of follow-up. High genetic diversity (12.5%) of P. vivax strains was recorded in the area. In general, relapse from P. vivax infection is responsible for the majority of recurrent malaria [68].

Recrudescence, with its potential to reveal treatment failure, carries an essential implication for the effectiveness of malaria treatment [5]. Recrudescence of P. falciparum 13 years after exposure was reported from Liberia. This woman is 49 years old with no history of traveling outside Canada, and never received a blood transfusion [69]. Recrudescence of P. falciparum after 4 years in a Ghanian pregnant woman living in Italy raised a speculation on the role of pregnancy triggering too late recrudescence by impairing preexisting immunity. Additionally, after relocating from a malaria-endemic area to a nonendemic area, patients may lose their immunity and develop recrudescence of the chronic P. falciparum infection [70].

4.1.2 Sub-Saharan Africa

Given the quality of interventions, co-endemicity of parasites, and climatic conditions, recurrence remains a formidable problem in sub-Saharan Africa [71]. Recrudescence of P. falciparum 13 years after exposure of a woman was reported in Liberia. This woman was 49 years old with no history of traveling outside Canada and never received a blood transfusion [69]. In Malawi, nearly 30% of children treated for malaria are reinfected within 42 days of treatment [72]. Greater immunity to P. vivax was conferred by reinfection with homologous parasite strain than with heterologous parasite strain. This was indicated by a reduction in the geometric mean of parasite count and in fever episodes from primary infection to reinfection [73]. These findings reflect the remaining problem in sub-Saharan Africa despite progress in the twenty-first century [74].

4.1.3 Ethiopia

Ethiopia as a sub-Saharan African country with a 75% malarious landmass, where P. falciparum and P. vivax are co-endemic, suffers from multidimensional burden of malaria and its recurrence. The risk of malaria recurrence in Ethiopia by day 28 and day 42 was 2.8% (95% CI: 0.9–8.4%) and 6.7% (95% CI: 3.2–13.5%), respectively [75]. The risk of P. vivax infection following treatment with rapidly eliminated treatments needs attention. After radical cure of primary P. vivax infection with PQ and CQ , 0.4 P. vivax infections/person/year was reported from Ethiopia by Abreha et al. [76]. Assuming these all as reinfections and the potential of slowly eliminated ACTs, the expected risk of P. vivax recurrence by day 63, 3.8% incidence is estimated in Ethiopia [12].

Malaria recurrence is among important public health problems in Ethiopia. An open-label trial aimed to assess the effectiveness of artemether lumefantrine (AL) in Jimma Zone by Eshetu et al., detected a 3.8% and 5.1% reinfection and recrudescence of P. falciparum by day 42, respectively. The same study identified a higher (9.4%) rate of P. falciparum recrudescence among children aged below 6 years in the Jimma zone. The lower capacity of children to sufficiently metabolize the drug has been suggested for the therapeutic yield [77]. Similarly, recurrent parasitemia rates of 19% and 7.5% after 28 days following treatment with AL and CQ , respectively, were observed in Ethiopia. This was considered as the incidence rate of treatment failure using in vivo parameters [78]. Furthermore, Teklehaimanot and his colleagues reported recurrent episodes of vivax malaria despite the use of an optimum dose of PQ and relocation of patients to a non-malarious area, from Gambella to Addis Ababa. This report alarms failure of PQ to prevent relapse in Ethiopia, where P. vivax is responsible for 40% of malaria prevalence (Figure 6) [79].

Figure 6.

Overview of malaria recurrence and its public health importance. Malaria recurrence can assume various forms depending upon the species of the plasmodium and other factors. (A) Primary infection due to inoculation of sporozoites by infected mosquito. (B) Sub-microscopic parasitemia is often missed by conventional diagnostic tools. (C) Infection with malaria parasites can be primary or recurrence. (D) Factors arising from the human host, the parasite, or intervention can determine the type of malaria recurrence. (E) Hypnozoites from the liver can give rise to recurrence or vice versa. (F) the prevalence of malaria recurrence depends on places, human hosts, type of drugs used, and season. (G) Recurrence is characterized by renewed parasitemia in peripheral circulation due to relapse, reinfection, or recrudescence. (H) Public health implication of malaria recurrence. Colored small circles: Green = new parasite injected as sporozoite, pale blue = hypnozoites from newly injected parasites, purple = existing parasites as sub-microscopic in the circulation, and red = long existing hypnozoite.

4.2 Factors affecting malaria recurrence

The recurrence of malaria depends upon various factors. These factors can be associated with the human host (gender, gene, and others), the Plasmodium parasite (specie, mono or mixed infection, and density), and intervention deployed (type of antimalarial drug) [12, 17, 80, 81, 82].

4.2.1 Human host factors

4.2.1.1 Genetic factors

The pattern of malaria recurrence, especially relapse, depends on host factors; including the cytochrome enzyme responsible for metabolizing anti-relapse drug. The prominent anti-relapse drug, PQ , is converted into its active metabolites in the liver by monoamine oxide and CYP2D6 (isotype of cytochrome P450 [CYP450]) enzymes [81]. CYP2D6 is naturally polymorphic, and its variant is widespread; up to 25% of the world’s population. This variant is associated with a substantial decline in efficacy of PQ putting people with this variant gene at higher risk of relapse [82, 83]. The host immune efficiency to clear parasitemia during the initial infection is important for the possibility of future recurrence [17]. The risk of P. vivax recurrence among patients who failed to clear their initial parasitemia within 2 days (AHR = 1.8 95% CI: 1.4–2.3; P < 0.001) [12]. People with a deficiency of the G-6-PD enzyme, owing to their ineligibility to PQ and TQ , carry a higher risk of vivax malaria recurrence [9, 61]. Pregnant and those breastfeeding children below 6 months, G-6-PD-deficient individuals, and children below 6 months do not receive PQ , and hence are at risk of recurrence [84, 85]. Also, according to a mathematical modeling study in Brazil, once infected with P. vivax, 23% of pregnant women are at risk of one or more episodes of vivax recurrence within 3 months [86]. Another modeling result shows the rise in P. vivax recurrence with an increase in the proportion of people with G-6-PD deficiency in the population [26].

4.2.1.2 Age

Being at a younger age raised the risk of P. vivax infection recurrence in the Thai-Myanmar border (P = 0.001) [63]. Patients with younger age (AHR = 3.04 95% CI: 2.39–3.87, P < 0.001) carry a greater risk of developing recurrent P. vivax parasitemia than adults [12]. Likewise, Antonio et al. 2021, recently reported a higher frequency of malaria recurrences among children under 4 years [87]. Slow clearance of parasites due to dose variation by age and weight; and subtherapeutic dose due to manipulation to overcome the bitter taste of drugs, particularly CQ , are some of the factors that are suggested to escalate the frequency of recurrence among children [87, 88, 89].

4.2.1.3 Gender

Male gender increased the risk of recurrence during the year in Brazil and Thailand [62, 63, 66]. Male patients (AHR = 1.26 95% CI: 1.08–1.46; P = 0.003) carry a greater risk of developing recurrent P. vivax [12]. This might be due to the behavior of the male that exposes him to a new infection with P. falciparum, which in turn triggers relapse. Additionally, more proportion of males is not eligible for PQ than females, which might raise the risk [12, 80, 90].

4.2.2 Parasite factor

Parasite-related factors, such as species, strain, density, and stage, play a fundamental role in the pattern of malaria recurrence [9].

4.2.2.1 Species

Species of the offending parasite are the leading factors driving malaria recurrence. P. vivax is responsible for the majority of recurrent malaria [9, 87]. A long-term study conducted on Thai-Myanmar border, revealed a higher frequency of P. vivax than P. falciparum recurrence, with a cumulative proportion of 31.5% (95% CI: 30.1–33.0%) and 21.5% (95% CI: 20.3–22.8%) recurrence by day 63, respectively [63]. Similarly, other supportive findings show the contribution of P. vivax for at least 70% of malaria recurrences [12, 62].

4.2.2.2 Density

The density of parasites, mainly the load of asexual parasites in the peripheral circulation enhances recurrence, particularly recrudescence, by giving resilience to the dose of treatment. Higher parasitemia and a shorter time since the onset of symptoms in the initial infection increased the risk of relapse during the year in Brazil and the Thai-Myanmar border [63, 66]. A similar effect of hyperparasitemia on severe recrudescence was observed in France, where a polyclonal infection and high load of parasites increased the risk in a participant, who returned from Chad [91]. On the other hand, patients with high parasite count (AHR = 1.59 95% CI: 1.22–2.08; P = 0.001), carry a greater risk of developing recurrent P. vivax [12]. Furthermore, according to a modeling study, individuals with more hypnozoites are predicted to experience more relapses [60].

4.2.2.3 Mixed-infection

In areas, where P. falciparum and P. vivax malaria are co-endemic, patients treated for P. falciparum infection have a high risk of subsequent P. vivax malaria. According to Commons et al., the risk of P. vivax parasitemia after P. falciparum at day 63 was 24% [12]. A series of RCTs in Thai-Myanmar revealed that a mixed infection and P. falciparum gametocytemia at enrollment raised the risk of P. vivax recurrence (P = 0.001) [63]. Similar other studies indicated the increased risk of relapse of P. vivax after infection with P. falciparum [80]. This suggests the potential role of P. falciparum infection to trigger the dormant P. vivax [12, 80]. Due to the suppression by P. falciparum, P. vivax disappears from the blood without reaching a density that can elicit a host immune response. Taking this defeat as a chance or tactic, P. vivax reemerges after weeks or months by generating transmissible densities of gametocytes. Besides, in low transmission settings, when immunity to P. falciparum is weak, falciparum malaria in adults may trigger dormant hypnozoites of P. vivax due to its capacity to trigger fever [20].

The growing pieces of evidence from all over the world about the correlation between the risks of P. vivax after P. falciparum treatment raise curiosity that the immune response of the host to acute malaria might trigger the revival of P. vivax hypnozoites. Besides, although the exact mechanism remains unclear, fever and hemolysis in P. falciparum infection are the suggested triggers for relapse [12, 80]. According to the report of the study in Thailand by Douglas et al., 51% of patients diagnosed with acute infection of P. falciparum and treated with a rapidly eliminated drug suffered a relapse of P. vivax after just 2 months [63]. Furthermore, a meta-analysis of clinical trials on P. falciparum by Commons et al. demonstrated that within 63 days, 24% of P. falciparum patients suffered from a P. vivax recurrence. This review reported that nearly 70% of malaria recurrences are due to P. vivax [12]. Taken together, these shreds of evidence not only show the sizeable input of relapse to the general epidemiology of malaria but also demonstrate how relapse wisely uses the inoculated sporozoites to optimize the survival of these species [11, 20].

4.2.3 Intervention-related and other factors

4.2.3.1 Drug

Drugs are profound determinants of malaria recurrence [63]. According to a series of clinical trials conducted for nearly 15 years on the Thai-Myanmar border, recurrence of P. vivax infection after 63 days was higher by 3.6 to 4.2-fold among participants treated with artemether-lumefantrine and artesunate-atovaquone-proguanil combinations compared to those treated with artemisinin-based combinations involving mefloquine or piperaquine [63].

ACTs containing mefloquine or piperaquine are better than ACTs containing AL at delaying the risk of recurrence. However, time-dependent efficacy decline has been revealed by a systematic review that reported a 15% prevalence of P. vivax recurrence among ACT recipients after day 63. The risk of P. vivax recurrence raises with the number of days after any antimalarial treatment given for P. falciparum infection [12]. Also, artesunate monotherapy may lead to recrudescence of parasitemia in 40–50% of cases within 28 days owing to drug-induced dormancy [92]. A comparable result has been observed in France, on a patient who returned from Chad [91].

Slowly eliminated antimalarials reduce the likelihood of early recurrence. Douglas et al. reported that the cumulative risk of P. vivax recurrence secondary to P. falciparum mono-infection was 51.1% after treatment with rapidly eliminated drugs (t1/2 < 1 day), 35.3% after treatment with intermediate half-life drugs (t1/2 1–7 days), and 19.6% after treatment with slowly eliminated drugs (t1/2 > 7 days) (P < 0.001) by day 63 [63]. Consistently, the risk of recurrence was higher for AL than for DHA-PPQ therapy. The proposed reason is the shorter dormancy of parasitemia for rapidly eliminated drugs, such as lumefantrine, in AL [12, 93].

Incorporating PQ , hypnozoiticidal drug, in standard malaria drugs reduces the risk of recurrence, particularly relapse. Primaquine, when combined with ACT and PQ , can reduce the risk of recurrence in P. vivax and P. falciparum co-endemic settings [12]. Consistent with this, PQ combined with either CQ or AL has reduced the recurrence of P. vivax infection among G-6-PD wild patients by 5-fold for 1 year in Ethiopia [76]. Considering a one-year-long RCT in Thailand, a high dose of PQ (7 mg/kg) over 7 or 14 days is efficacious in preventing P. vivax relapse. Similarly, at least one episode of P. vivax recurrence has occurred among 70% of subjects in non-PQ arms compared to 18% of subjects in the PQ arm [15].

According to a systematic review by Commons et al., the risk of P. vivax recurrence with a lower dose of CQ was 32.4% (95% CI: 29.8–35.1) by day 42. However, raising the dose substantially reduced the risk. Furthermore, including PQ in the treatment significantly reduced the risk of recurrence by day 42 from (AHR = 0.82 95% CI: 0.69–0.97; P = 0.021) with CQ alone to (AHR = 0.10, 0.05–0.17; P < 0.0001) with CQ and PQ. This strengthens the concept of the role of PQ as a radical cure in effectively preventing the early recurrence of P. vivax [89]. Nevertheless, despite the renowned efficacy of PQ , few pieces of evidence are emerging on malaria recurrence after radical therapy. Considering the tests, they used microscopy and the unknown status of CYP2D6, these pieces of evidence reflect treatment failure, but do not confirm drug resistance [79]. After its introduction, ACT rendered the rate of recrudescence to be below 10% in Jimma zone, southwest Ethiopia [77]. It is strongly considered that the probability of artemisinin resistance with a standard dose, a 3 day ACT, is rare if the proportion of day 3 positive smears is below 3% [94].

4.2.3.2 Place

In co-endemic locations, patients presenting with P. falciparum are highly likely to carry P. vivax hypnozoites that give rise to recurrence [12, 80]. Patients in regions of short relapse periodicity had a greater rate of P. vivax parasitemia than those in regions of long periodicity (HR = 8.61 95% CI: 2.34–31.65; P = 0.001) [12]. Also, the risk of P. vivax parasitemia was 6.5% (95% CI: 4.6–8.6) in regions of short periodicity compared with 1.9% (95% CI: 0.4–4.0) in areas of long periodicity [12, 17]. Therefore, geographical variation determines the frequency of malaria recurrence.

4.2.3.3 Season

A dormant hypnozoite of P. vivax awaits the right time for activation to take evolutionary advantage, thereby ensuring maximum likelihood of transmission and immune evasion. To ensure optimum transmission, hypnozoites synchronize their wake-up time to a season with an abundant mosquito population [20]. Thus, the incidence of symptomatic malaria was higher in September (OR = 2.81 95% CI: 2.1–3.7) and October (OR = 2.4 95% CI: 1.8–3.2) than in November [95].

5. Recurrence and malaria transmission

Malaria recurrence, in addition to rising clinical episodes, increases the probability of malaria transmission. In fact, all types of malaria recurrence increase transmission in multidimensional ways, including by raising longevity of infectiousness and transmission potential [6, 11, 20]. A systematic review and meta-analysis result revealed that recrudescent (AOR = 9.05 95% CI: 3.74–21.9) and reinfection (AOR = 3.03 95% CI: 1.66–5.54) with P. falciparum were strongly associated with gametocytemia after day 7 [29]. Hence, recurrent infections with Plasmodium species maintain the potential for malaria transmission [6, 9, 16].

Recurring malaria leads to erythrocytic schizogony that renews or escalates peripheral parasitemia. Such parasitemia entails gametocytogenesis that maintains the transmission of malaria, thereby complicating elimination and eradication efforts [6, 16, 96, 97]. Moreover, activation of hypnozoites from different earlier inoculations can produce at least two genotypes simultaneously growing inside the patient’s blood. This produces genetically distinct gametocytes that, if taken by a mosquito, have a high probability to undergo meiotic recombination resulting in genetic variation. This phenomenon is considered as a major factor for high degree of genetic diversity seen in P. vivax over settings with very low seasonal transmission [17]. Hence, recurrence confers P. vivax with maximum opportunity for transmission and immune evasion [20].

Recurrence of P. vivax worsens transmission of malaria with its potential to promptly produce gametocytes and transmit before treatment initiation. In addition, due to the continued reactivation of hypnozoite, the patient remains a potential reservoir of infection for a long time from just a single inoculum [9, 97]. The probability of relapse and subsequent transmission raises with a load of hypnozoite in the liver [6, 60]. Furthermore, if the recurrence involves a heterologous genotype, it paves the ways for the development of drug resistance, thereby transmission of variant species. The variant species together with other factors cause recrudescence, which expands the pool of recurrence, and the subsequent transmission [20]. Besides, reinfection further exacerbates transmission dynamics with its potential to activate hypnozoite; introduce a new strain unresponsive to therapy and provides an opportunity for immune evasion [11].

Generally, malaria recurrence sustains and/or intensifies transmission. It raises efficiency, chance, and longevity of transmission, thus threatening the success of interventions [11]. Thus, it is wise to consider fighting recurrence to break transmission and eliminate malaria (Figure 7) [60, 96, 97, 98].

Figure 7.

Pattern of malaria recurrence, spots to intervene, and its prevention strategies. Parasitemia in peripheral circulation can be due to symptomatic, asymptomatic, or sub-microscopic plasmodium infection. This peripheral parasitemia can also arise from any type of malaria recurrence. (red boxes): After arriving in the liver, the inoculated sporozoites either transform into schizonts or directly become hypnozoites that later can become schizonts. Schizonts arising from latent hypnozoites or the inoculated sporozoites release merozoites that join the blood circulation to eventually produce asexual parasites. (purple boxes and lines): Young gametocytes localize into the hematopoietic niche and rejoin the circulation when reach stage five gametocyte. Gametocytes can result from unsuccessful treatment, after successful treatment with non-gametocidal drugs, and any untreated infection. These gametocytes mediate the transmission of malaria. (broken blue lines): Asexual parasites that periodically sequester in the hematopoietic niche and return into circulation resulting in relapse-like parasitemia. (blue solid lines): Flow of malaria pathogenesis from primary and reinfection subsequent to inoculation of sporozoites. (black line): Parasites sequestering in the hematopoietic niche. (greenish-yellow box and lines): Unsuccessful treatment and sub-microscopic infections give rise to recrudescence and/or possible transmission. (Green lines): Possible weak points for intervention (such as prompt diagnosis and effective treatment with efficacious radical cure, and transmission-blocker) to tackle recurrence and/or transmission.

6. Recurrence and malaria elimination strategies

The axiom “prevention is better than cure” better describes the impact of recurrence on the efforts to eliminate malaria [9, 85]. The difficulty to diagnose and treat malaria substantially increases morbidity and mortality among patients. Relapse due to vivax malaria is refractory to the majority of the current treatments since the sole hypnozoiticidal-drug, PQ , is challenged by many factors [9, 20]. The use of ACT will more noticeably reduce the prevalence of P. falciparum prevalence than P. vivax due to its inability to kill the hypnozoite forms of P. vivax [19].

A relapse followed by asymptomatic parasitemia could be the major approach to P. vivax transmission [20]. Particularly recurrence from asymptomatic infections is the major challenge to the elimination of malaria as they arrive at the gametocyte stage at the level below the sensitivity of the current gold standard tool for malaria diagnosis: light microscopy [9, 17]. Asymptomatic P. vivax infections are common in malaria-endemic locations [36]. However, the contribution of relapse for asymptomatic malaria is not well known [9, 16, 19].

Vivax malaria remained the major global challenge despite a spectacular achievement against P. falciparum [74]. The complex nature of P. vivax, mainly due to relapse, extensively wrinkled the effectiveness of the radical cure with PQ. A considerable portion of the global population has difficulty converting PQ into its active form, hence the increased risk of recurrence [9]. Pregnant women and children below 6 months old owing to their ineligibility to PQ are at risk of vivax malaria and its recurrence. They can also serve as potential human reservoirs of infection [9, 85]. Hence, it is prevention that helps to keep this ever-complicating parasite at its bay [9, 16, 17, 99].

The possibility of relapse secondary to treatment and/or immunologic response to P. falciparum infection perpetuates the transmission in co-endemic settings. The growing phenomena of resistance to antimalarials, such as CQ and ACT, is another fuel to recurrence. The ineligibility of the vast majority of population to PQ given their physiological and genetic condition places a hurdle on the stride toward preventing recurrence [9].

The frequent possibility of mutation in P. vivax owing to cross-reactivity between strain arising from hypnozoite and strain from mosquito in an environment challenges the efficacy of therapies [14, 17]. Different intervals of recurrence resulting from the variation of P. vivax population in tropics (frequent relapse) and temperate (long latency) regions is another nightmare. Moreover, the capacity to remain sub-clinical and submicroscopic with huge potential to quickly produce gametocytes offers the cutting-edge to this parasite triumph [11, 17, 20, 100].

In general, the recurrence of Plasmodium infection, especially the P. vivax relapse, is the major setback to malaria elimination strategies of the time. Consequently, there is a renewed focus on P. vivax due to the mounting accumulation of evidence on the maliciousness of vivax malaria [9]. To achieve malaria eradication goal, we must consider all species of Plasmodium; while improving the transmission-reducing potential of interventions [6, 14, 19]. It is key to underpin prevention mainly for infants and pregnant women, while keeping the search for targets of intervention to stay in the game [9].

7. Conclusion and future remarks

Malaria recurrence plays a considerable part in sustaining malaria epidemiology. Understanding the biology and epidemiology of malaria recurrence is crucial to overcome current challenges to efforts in malaria elimination. Malaria recurrence is not adequately studied partly due to the complex nature of the disease and logistic constraints for diagnosis. Regardless of its role in complicating clinical and public health impact of malaria, a basic understanding of malaria recurrence is limited. In this review, we have discussed the biology and epidemiology of malaria recurrence along with its implication on malaria mitigation.

Recurring malaria leads to increased morbidity and mortality due to malaria. By creating opportunities for gametocytogenesis, it sustains malaria transmission. Without identifying the specific Plasmodium species responsible for recurrence in specific areas limits the effectiveness of interventions. Lack of clear idea about the specific type of recurrence raises misconception on drug efficacy that eventually leads to hasty prohibition of antimalarials without robust replacement. Without effective intervention based on a clear understanding of recurrent malaria, morbidity and mortality continue to escalate due to recurrence. Unmanaged recurrent malaria maintains transmission of malaria. Hence, failure to promptly deal with recurrent malaria in a certain area has a huge impact on the health of individuals and the community at large.

Due to its widespread prevalence, relapse propensity, complex process of diagnosis and treatment, as well as other aspects, P. vivax demands continued multidimensional research. Studies should focus on the biology of malaria recurrence to improve the yield of control interventions. Optimizing disease-control interventions prevent a recurrence and breaks transmission to eliminate malaria.

8. Outstanding questions

1. How to detect and quantify P. vivax hypnozoite?

2. How much is the contribution of relapse for asymptomatic malaria?

3. How to detect and identify markers to characterize three types of recurrence in vivax malaria?

4. How to differentiate non-hypnozoite-driven and non-bloodstream-originating relapse?

5. How can we measure the contribution of the hematopoietic niche to malaria recurrence?

6. What is the frequency of malaria recurrence among the “PQ-hard-to-reach population,” particularly in settings where P. falciparum and P. vivax are co-endemic?

7. How can we measure/model the contribution of the “PQ-hard-to-reach populations” to the overall transmission potential?

8. How can we improve the prevention and control of malaria among the most vulnerable, but “PQ-hard-to-reach population?”

9. After how many recurrences will a new drug-resistant strain/species develop? and does it depend on species?

10. How much of reinfection with P. vivax will remain asymptomatic in mixed infection (P. falciparum + P. vivax)?

11. What is the pattern of recurrence by mono-infection and mixed-infection?

12. What proportion of mixed infection (P. falciparum + P. vivax) is properly treated and/or miss-treated as P. falciparum single infection?

13. How can we differentiate non-hypnozoite-originating relapse from true relapse?

9. Search strategy and selection criteria

We searched PubMed, the Cochrane Library, Science Direct, and Google Scholar for articles published in English between January 01, 2000, and December 31, 2021, with the terms “malaria recurrence,” “malaria relapse,” “malaria reinfection,” “Plasmodium vivax,” “recurrent parasitemia,” “malaria recrudescence” and combined with the terms “glucose-six-phosphate dehydrogenase, “primaquine.” We also included review studies (published between January 01, 2000, and December 31, 2021) cited by articles identified by this search strategy and selected those we identified as relevant. Selected review articles are cited to provide readers with more details and references than this review can accommodate.

Acknowledgments

The author is grateful to Mr. Wasihun Admassu for his important comments on the first draft of this paper. The author also thanks Mr. Ahmed Kamil for his help in the drawing process of diagrams.

Author’s contributions

AA has conceived and designed this review. AA has participated in the acquisition of data and preparation of the draft manuscript. AA is involved in drafting, critically reviewing, and final approval of this manuscript.

Abbreviations

ACT	artemisinin combination therapy
AHR	adjusted hazard ratio
AL	artemether lumefantrine
CI	confidence interval
CQ	chloroquine
CYP450	cytochrome P450
DNA	de-oxy ribonucleic acid
DHA-PPQ	dihydro-artimesinine piperaquine
GLURP	glutamate rich protein
G-6-PD	glucose six phosphate dehydrogenase
MSP	merozoite surface protein
nPCR	nested polymerase chain reaction
OR	odds ratio
PCR	polymerase chain reaction
PQ	premaquine
qPCR	quantitative polymerase chain reaction
rRNA	ribosomal ribonucleic acid
TQ	tafenoquine