Background
Sri Lanka eliminated indigenous malaria in 2012, and was certified as a malaria free country in 2016. The conducive tropical environment still facilitates the perennial breeding of the major vector of malaria,
Anopheles culicifacies, in many parts of the country [
1]. Therefore, the country is at high risk of re-establishment of the disease [
2]. This was aptly demonstrated by the introduced malaria case diagnosed in 2018 [
3]. The introduction of the urban invasive vector species
Anopheles stephensi a few years ago has increased receptivity [
4]. The Anti Malaria Campaign (AMC), in line with the national strategic plan for malaria, is taking measures to prevent the re-establishment of malaria and prevent deaths due to malaria [
5]. As a requirement, quality-assured diagnostic service is maintained in the country. Passive case detection is carried out comprehensively. In addition, the AMC is conducting targeted proactive case detection among clusters of high-risk individuals based on the importation risk [
6]. Currently, approximately 50 imported malaria cases are reported each year [
7]. Each reported case is fully investigated and followed up according to integrated drug efficacy surveillance (iDES) [
8,
9]. Reactive parasitological and entomological surveillance is conducted based on the history of travel and the possibility of local transmission [
10]. The findings are reviewed by an expert panel, the Case Review Committee of the AMC before the case is classified as imported, indigenous, introduced, relapse, or recrudescence [
11]. As a country in the POR phase any recurrent infection classified as a reinfection, in a patient that has no history of overseas travel to a malaria-endemic country, would be an introduced case or an indigenous case. As this means the resumption of local transmission, comprehensive interventions are needed to be done immediately. If a recurrent infection is classified as a recrudescence, the possibility of treatment failure needs to be considered, and there is a need to re-visit the anti-malarial treatment guidelines. Although reactive surveillance often provides the answer, this requires a lot of field activities and resources. Occasionally, that alone is not adequate to conclude the case classification with certainty, especially when there is a long duration between the initial and recurrent infection.
Genotyping in identifying introduced and indigenous malaria cases in countries that have eliminated malaria has been well documented [
12]. To distinguish recrudescence (true treatment failure) from a reinfection of
Plasmodium falciparum, a cost-effective PCR genotyping protocol has been recommended by the World Health Organization (WHO) and Medicines for Malaria Venture (MMV) [
13,
14]. Based on length polymorphic genes encoding the merozoite surface proteins (
msp1 and
msp2) and the glutamate-rich protein (
glurp), this enabled application of genotyping even in settings with limited resources. Recently, this method has been criticized as underestimating true drug failure rates in certain epidemiological conditions [
15‐
18] and the WHO has published revised guidelines recommending the use of microsatellites instead of
glurp for low to moderate and high transmission settings in Africa, while outside Africa the previous protocol of the
msp1, msp2 and
glurp genotyping is still applicable [
19]. As a country in the POR phase with imported malaria cases originating from countries with varying endemicities, any misleading interpretation would have a detrimental impact on the malaria-free status of Sri Lanka. This study assessed the relevance and the role of the
msp1, msp2 and
glurp genotyping procedure to differentiate
P. falciparum recrudescence from reinfections in the POR phase in Sri Lanka.
Discussion
As a country that has eliminated malaria, Sri Lanka needs to prevent re-establishment of malaria transmission in the country. This requires immediate preventive measures for any imported, introduced, or indigenous case. Considering the P. falciparum artemisinin partial resistance, immediate identification of recrudescence from reinfections is crucial.
Unlike in therapeutic efficacy studies in endemic settings, where “molecular correction” by genotyping is used to guide the use of anti-malarials, obtaining evidence for the malaria-free status was an objective of this study. Hence, the genotyping outcome was compared with epidemiological findings. The six patients with recurrent infections had a history of travel to African countries. Genotyping revealed highly polymorphic genotypes in primary infections of these recurrent infections, indicating that they have been initially exposed to high transmission settings. Heavy multiple infectious bites per person are common in sub–Saharan African countries with high transmission intensity [
23,
24]. Thus, the African origin of these infections with recurrent attacks correlates well with the genotyping findings.
PCR is used to differentiate between reinfection and recrudescence by comparing the allelic variants present in the initial and recurrent samples. In high transmission settings, when patients harbour high multiplicity of infections or common genotypes, it may be difficult to differentiate reinfections from recrudescence. In such settings,
P. falciparum reinfections as early as day 14 are known to occur [
25]. Other studies have shown persisting asexual parasitaemia with more than 25% prevalence even after 14 days [
26]. Depending on the prevalence of allelic variants in a particular region, there is always a possibility of a reinfection with the same genotypes that are in the patient before treatment. With the recent recommendations for advanced genotyping procedures for African countries [
19], it will be important to determine whether the genotyping protocol used in this study would relate to the epidemiological findings in differentiating recrudescence from reinfections and thereby provide evidence to maintain malaria-free status.
Methodologies used for genotyping
P. falciparum have their advantages and limitations. Capillary electrophoresis (CE) has been recommended for precise fragment sizing for genotyping especially in high transmission settings [
27]. However, CE may not be readily available in resource-limited settings. In such situations, the limitations of the
msp1, msp2 and
glurp genotyping procedure need to be properly assessed especially when applying the findings for decision-making.
This study was done in a setting where local transmission has been eliminated and selective control measures were continued in the malaria POR phase. Since the resources were limited, agarose gel electrophoresis was performed and fragment sizes were determined manually which resulted in five recrudescence and one reinfection. It is known that the limited resolution of this method (where genotypes differing in less than 20 bp for
mps1 and
msp2 and less than 50 bp for
glurp are considered as one) can be a major factor in the overestimation of the treatment failure rate or in some instances mis-classifying recrudescences as new infection [
15,
28,
29]. However, the findings of this study compared well with the epidemiological findings indicating the applicability of genotying protocol used.
Yet, the limitations of this genotyping procedure need to be properly identified and assessed. Amplification bias is known to suppress long fragment alleles and preferentially amplify short fragments thereby compromising the detection of co-infecting clones [
17]. In high transmission settings, the range of MOI has been reported from a single strain to more than 10 strains in an isolate. In such multi-clonal infections, in the presence of a dominant clone, the minority clones consisting a small proportion of biomass might fall below the detection limit of the genotyping method. Such cryptic minor variants can be missed by PCR- based detection due to completion for primer or other constituents of the reaction mix by the more abundant clones that may be present in a patient blood [
15‐
17,
28‐
33]. Due to this imperfect clone detectability, variants that would be missed in the initial genotyping would later appear at a detectable level in the recurrent parasitaemia (resistant) and there is a possibility of misidentifying such a true recrudescence as a false reinfection. In this study too, the observation of one or more extra alleles of
msp1 and
msp2 genotyping in post-treatment samples in some of the patients is a good example for this phenomenon. However, as was evident in this study, the WHO definition of a recrudescence, that is the presence of at least one shared genotype in the compared pre and post-treatment samples at all loci seems to be an effective method that would overcome most of the above limitations [
13]. In the absence of local transmission, (as confirmed by the reactive entomological and parasitological surveillance) this confirms that the infections are actual recrudescences. This highlights the accuracy of
msp1, msp2 and
glurp genotyping method in categorizing recurrent infections of imported malaria cases in the POR phase in Sri Lanka. Furthermore, the outcome of genotyping confirmed the epidemiological finding which indicated that these patients have not travelled out of the country in between the initial and recurrent infection and that there was no evidence for local transmission.
Persisting parasitaemia after 42 days post-treatment have been reported among imported malaria cases in malaria-free settings [
34]. For countries with high malariogenic potential such as Sri Lanka, such persisting parasitaemia and recrudescence would result in extensive reactive surveillance and control measures. Therefore, it would be important to find out with certainty whether such a recurrent infection is a recrudescence or a reinfection. In fact, the WHO recommends genotyping to confirm all reinfections and recrudescence that occur after 28 days [
35]. In this study, genotyping confirmed that the recurrent infection detected after a lapse of 3 months (105 days) was a reinfection. Unlike in malaria endemic countries in the POR phase with high malariogenic potential, it is important to find out whether this reinfection is due to local transmission or due to a recent travel to a malaria endemic country. Since either genotyping or epidemiological findings alone may not provide conclusive evidence, combining the genotyping outcome with epidemiological findings will help to determine the origin of the infection.
According to the
msp1, msp2 and
glurp genotyping procedure, if any marker shows only new alleles, the recurrent infection is considered a reinfection. This algorithm had been criticized as it had consistently underestimated true failure rates [
17,
29] and obtaining consensus of two genotypes (2/3 algorithm) has been suggested by some researchers [
27]. For the recrudescences analysed in this study, both methods would have given the same result. These criticisms were mainly due to the unreliable nature of
glurp. This was also seen in this study, where
msp1 and
msp2 markers performed well, but
glurp genotyping failed even after repeated testing in 50% of the samples. It may be assumed that the cause for this poor performance may not be an issue of the DNA template, or low copy number in the initial samples since
glurp was not amplified even in samples with high parasitaemia, and when other genes have been amplified. Also other intrinsic factors could be a cause for this non amplification. Considering the unreliable nature of
glurp, the WHO has recommended replacing
glurp with a microsatellite marker for low to moderate and high transmission settings in Africa. For countries outside Africa, the current method is still applicable [
19].
The information on the drug efficacy of antimalarials available in countries like Sri Lanka, where treatment outcomes can be observed without repeat inoculations from infectious mosquitoes would be useful for the status of drug resistance in malaria-endemic countries. However, it is important to note that treatment failure due to resistance to the ACT is only one of the possible reasons for recrudescence. Other possibilities like defects in absorbance and metabolism of drugs also need to be considered [
36].
With zero indigenous disease burden, prompt case detection by health institutions, and healthcare providers is a major challenge as malaria is low in the differential diagnosis of patients presenting with fever. In some instances, this has resulted in unacceptable delays in diagnosis, sometimes even exceeding 30 days since the onset of fever [
7]. In addition to the harmful effect on the patient, there is always a possibility that such a delay can result in the re-establishment of local transmission in Sri Lanka. This requires reactive surveillance to be carried out especially where the risk of importation and receptivity is high. Obtaining confirmatory evidence for the absence of local transmission as seen for these recurrent infections is important. In this study, the genotypically confirmed reinfection had a history of traveling to a malaria-endemic country in between the two infections. This indicates that the reinfection was contracted in that country. Since reinfection without a history of travel to a malaria-endemic country would mean local transmission, this highlights the importance of combining the genotyping outcome with the findings of the case investigation and reactive surveillance to ensure the malaria-free status.
In this study, the genotyping of initial and recurrent infections were performed when a recurrent infection was detected. Therefore all PCR assays were performed before revision of the WHO guidance in 2021. Since even according to the revised guidelines, msp1, msp2 and glurp genotyping is applicable for countries outside Africa the findings of this study may be a good example for countries eliminating malaria or in the POR phase. In this context it is important to note that that msp1, msp2 and glurp genotyping protocol can be applied even when resources are limited as it does not require expensive equipment and is not labour intensive.
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