Given the ongoing challenges associated with the elimination of this serious disease, the current study investigated the frequency and distribution of pfcrt gene point mutations in P. falciparum isolates collected from Jazan region in southwestern Saudi Arabia, just over a decade after the cessation of CQ use and the adoption of ACT for the treatment of uncomplicated falciparum malaria.
The results revealed that the pfcrt 76T point mutation (the most important mutation linked with CQ resistance) was present in about two thirds (66.4%) of the examined isolates, whereas about one third (33.6%) of the isolates were found to carry the K76 wild variant. The results also showed a higher percentage of the wild-type K76 allele of the pfcrt gene in isolates from Saudi (41.2%) as compared to those from non-Saudi (29.7%) patients, and importantly, there were also some locally transmitted isolates (autochthonous malaria). Therefore, these results indicate the potential return of CQ-sensitive strains in Saudi Arabia.
Although CQ resistance and clinical treatment failures in the country were reported in the early 1990s (Malik et al. 1997), molecular studies on pfcrt were not conducted until in the mid-2000s. Prior to those studies, the CQ treatment failure rates varied between 12% and 38% (Alrajhi et al. 1999; Malik et al. 1998; Ghalib et al. 2001). The first molecular study on pfcrt gene polymorphism was conducted in 2007 and showed that about 90% of 19 P. falciparum isolates from Jazan region carried the pfcrt 76T mutated allele (Al Harithi et al. 2007). Later, other studies found the 76T mutated allele of pfcrt in 100% of 121 P. falciparum isolates from both the Jazan and Aseer regions (Bin Dajem and Al-Qahtani 2010; Bin Dajem et al. 2011). Likewise, a comprehensive previous study found that 99% of 165 P. falciparum isolates from Jazan were carrying the pfcrt 76T mutated allele (Bin Dajem et al. 2012). A more recent study detected the pfcrt 76T mutation in only four out of 13 (30.7%) P. falciparum isolates from Taif province, about 720 km north of Jazan, while the remainder (eight isolates) were carrying the wild allele (Soliman et al. 2018). However, the results of that study are limited by the very small sample size.
Overall, the withdrawal of CQ and the adoption of ACT treatments in Saudi Arabia in 2007 might explain the re-emergence of CQ-sensitive isolates, as identified by the current study. Yet, there is a dearth of information on the frequency of pfcrt point mutations in Saudi Arabia. Therefore, the hypothesis proposed herein that CQ-sensitive strains have reappeared in the country requires further evaluation. Moreover, microsatellite loci analyses are essential to compare the genetic similarity among CQ-susceptible and among CQ-resistant parasites as well as between parasite populations circulating before and after the official banning of CQ as a falciparum malaria chemotherapy in Saudi Arabia.
Interestingly, some previous studies, exclusively from Africa, have demonstrated the return of CQ-sensitive P. falciparum strains several years after the cessation of CQ use and have suggested that CQ might once again be effective. In 1994, Malawi was the first country in Africa to withdraw CQ, and 12 years later molecular markers of CQ resistance had completely disappeared and the K76 wild type had reached fixation (Kublin et al. 2003; Frosch et al. 2014). Subsequently, a randomised clinical trial demonstrated the superior efficacy of CQ against uncomplicated falciparum malaria in Malawi, with a cumulative efficacy of 99% as compared to 21% for SP, and also detected the K76 wild type in all isolates (Laufer et al. 2006). Similarly, in 1998, Kenya scaled up its national malaria drug policy and switched from CQ to SP. Subsequently, a study showed increasing rates of the K76 wild type until 2006 (Mwai et al. 2009). However, another study conducted in western Kenya, where CQ use continued despite a national-level policy change, showed that the prevalence of mutant type pfcrt 76T increased significantly, from 76% in 2001 to 94% in 2007 (Shah et al. 2015). Moreover, the re-emergence of CQ-sensitive falciparum malaria has been reported in other African countries, including Sudan (Bakhiet et al. 2019), Tanzania (Alifrangis et al. 2009; Mohammed et al. 2013), Côte d’Ivoire (Dagnogo et al. 2018) and Uganda (Balikagala et al. 2020).
Looking at those countries geographically closer to Saudi Arabia, the prevalence of the mutant pfcrt 76T allele in the neighbouring endemic country of Yemen was reported to be as high as 100% several years after switching from CQ to ACT in 2009 (Al-Mekhlafi et al. 2011; Alareqi et al. 2016; Atroosh et al. 2016). Similar findings were reported for Pakistan (Khattak et al. 2013; Khan et al. 2020). These findings are consistent with those of the current study, which showed that the highest percentage of the mutant pfcrt 76T allele was in the isolates from Yemeni (94.2%) followed by Pakistani (65.5%) patients, as compared to those from other nationalities. Bearing in mind that over 12 million foreign nationals are employed in Saudi Arabia, and that the majority of them are from malaria-endemic countries such as Pakistan, Yemen, Sudan and Ethiopia (US-SABC 2020), and that malaria in Jazan region is mostly imported, with a very low number of locally transmitted cases reported (Al-Mekhlafi et al. 2021), it would be desirable to carry out further studies on a larger number of samples to carefully evaluate pfcrt polymorphism according to nationality and source of case.
The findings of the current study also showed that the percentage of CVIET triple-mutant haplotype in the studied isolates was 56.2%, while about only one third (33.6%) of the isolates harboured the CVMNK wild haplotype. The only previous study that reported the presence of pfcrt-related haplotypes in Saudi Arabia was conducted in Jazan region and showed that almost all the studied isolates (99%; 163/165) harboured the triple-mutant CVIET haplotype (Ben Dajem et al. 2012). The reduction in the prevalence of this haplotype in the region is attributed to the re-emergence of the pfcrt K76 wild allele reported by the current study. Similar findings have also been reported in some African countries that witnessed a partial return of the pfcrt K76 wild allele (Alifrangis et al. 2009). The CVIET haplotype which originated in Southeast Asia is the most common haplotype in some African and Middle Eastern countries, including Yemen (Al-Hamidhi et al. 2013), and Sudan (Gadalla et al. 2010), whereas the SVMNT double mutant haplotype has remained the predominant haplotype in Asian countries close to the Arabian Peninsula, such as Pakistan (Sahar et al. 2015) and Iran (Ursing et al. 2006). These findings are consistent with the current study which found that the SVMNT haplotype was present only in isolates from Pakistani patients, while the percentage of the CVIET haplotype was highest in isolates from Yemeni patients.
The current study also examined the associations of point mutations and haplotypes in the pfcrt gene with the participants’ demographic factors and parasitaemia. The results showed that the percentage of the pfcrt 76T mutation and the percentage of the CVMNK wild haplotype was significantly higher in isolates from participants aged ≥ 30 years and < 30 years, respectively. The association between the pfcrt 76T mutation and the host’s age is controversial, where some studies have showed a significant association (May and Meyer 2003; Happi et al. 2006; Al-Mekhlafi et al. 2011) and others have reported no association (Atroosh et al. 2016; Acharya et al. 2018).
As regards a link with parasite density, the current study found that the percentages of the pfcrt 74I and 75E mutations and the CVIET mutant haplotype were significantly high in isolates from patients with a low parasitaemia level. Although the reason for these associations is unclear, previous studies reported significant associations of pfcrt mutations (particularly 76T) with parasitaemia and severity of infection (Al-Mekhlafi et al. 2011; Wélé et al. 2011; Atroosh et al. 2012; Acharya et al. 2018; Cuu et al. 2020). However, another study found no significant association (Mayeng et al. 2007; Atroosh et al. 2016).
In respect of a connection with residency, there was significant variation in the distribution of pfcrt polymorphism across the governorates involved in the current study. Almost all of the isolates from Aldair governorate were found to carry the mutant pfcrt 76T allele and over 90% of them were carrying the CVIET haplotype. Aldair borders Yemen to the south, and the highest prevalence of these markers was found in isolates from Yemeni patients. Studies conducted in Yemen have reported a very high prevalence of the pfcrt 76T mutation, which has reached fixation (100%) in some areas (Al-Mekhlafi et al. 2011; Alareqi et al. 2016; Atroosh et al. 2016). Therefore, taking into account that Jazan region is the smallest region of Saudi Arabia, the reported variation can be attributed to the patients’ residency. Nonetheless, the distinct variation in pfcrt 76T either between countries or within the same country has previously been reported (Bamaga et al. 2015; Shah et al. 2015).
When interpreting the findings of the current study that have been discussed above, it is important to take bear in mind that the study has a few limitations. First, only codons 72 to 76 of the pfcrt gene were assessed, while other codons such as 97, 220, 271, 326, 353, 356, and 371 were not included. Indeed, point mutation 76T has become a hallmark of CQ resistance worldwide, and is widely used as an epidemiological tool for the monitoring of CQ resistance in large-scale field studies (Djimdé et al. 2001; Roux et al. 2021). Moreover, the cluster of mutations at codons 72 to 76 describes the evolution of the main pfcrt genotypes, including the SVMNT ancestral wild haplotype as well as the related mutated haplotypes such as the predominant and widespread CVIET haplotype (Awasthi et al. 2012). Second, a small number of blood samples was collected from female as compared to male participants as well as from some governorates and some nationalities. Nonetheless, the current study still provides important data about the distribution of pfcrt polymorphism in Jazan region after over a decade of CQ treatment withdrawal.