The return of chloroquine-sensitive Plasmodium falciparum parasites in Jazan region, southwestern Saudi Arabia over a decade after the adoption of artemisinin-based combination therapy: analysis of genetic mutations in the pfcrt gene

This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11 years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76 T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified as follows: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%) and CVMNT single-allele (8.5%) mutant haplotypes and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate and nationality of the patients as well as the parasitaemia level (p < 0.05). The findings provide evidence of the potential re-emergence of CQ-susceptible P. falciparum strains in Jazan region over a decade after CQ discontinuation, with about one third of the isolates analysed carrying the pfcrt K76 CQ-sensitive wild allele and the CVMNK ancestral wild haplotype. Although the reintroduction of CQ cannot be recommended at present in Saudi Arabia, these findings support the rationale for a potential future role for CQ in malaria treatment. Therefore, continuous molecular and in vitro monitoring mutations of pfcrt polymorphism in Jazan region is highly recommended.


Introduction
Malaria, a mosquito-borne disease transmitted by the bite of the female Anopheles mosquito, is a leading cause of morbidity and mortality worldwide, especially in the tropics and Section Editor: Kevin S.W. Tan * Hesham M. Al-Mekhlafi halmekhlafi@yahoo.com subtropics. Despite global success in reducing the malaria burden over the last several decades and the successful elimination of malaria from more than 100 countries, the disease is still endemic in 106 countries (WHO 2020;Feachem et al. 2019). Antimalarial drug resistance has emerged as one of the most critical threats hampering the global efforts to control and eliminate malaria. For many decades, chloroquine (CQ) was used extensively as the first-line treatment for uncomplicated malaria. However, in the late 1950s, CQresistant Plasmodium falciparum malaria emerged independently in Southeast Asia (Thai-Cambodian border) and in South America (Colombia) (Payne 1987). Subsequently, resistant P. falciparum strains spread steadily across different countries throughout Southeast Asia and into India, as well as across South America in the 1960s and 1970s, and then spread to Africa in the late 1970s with confirmed treatment failures reported in Kenya and Tanzania (Fogh et al. 1979;Wellems and Plowe 2001). Yet, despite the increasing spread of CQ resistance worldwide, CQ remained the firstline treatment for malaria until the 2000s. It is well documented that CQ resistance is associated with specific mutations in polymorphism of the P. falciparum chloroquine resistance transporter (pfcrt) gene located on chromosome 7 (Roux et al. 2021). The cluster of mutations at codons 72 to 76 of the pfcrt gene has been found to be associated with the evolution of three main distinct haplotypes/genotypes: (1) the ancestral CQ-susceptible haplotype, which is found in CQ-sensitive isolates and referred to as C 72 V 73 M 74 N 75 K 76 ; (2) the double mutated haplotype S 72 V 73 M 74 N 75 T 76 , which is widely prevalent in Latin America and Asia; and (3) the triple-mutated haplotype C 72 V 73 I 74 E 75 T 76 , which predominates in Africa and Southeast Asia (mutated alleles are shown in bold) (Ariey et al. 2006;Awasthi et al. 2012). Moreover, other mutated haplotypes such as C 72 V 73 M 74 E 74 T 76 , C 72 V 73 M 74 N 75 T 76 and S 72 V 73 I 74 E 75 T 76 have been reported in other regions (Awasthi et al. 2012;Atroosh et al. 2016). Studies have also revealed that a point mutation at codon 76 of the pfcrt gene, which occurs as a result of lysine amino acid substitution by threonine (wild-type K to mutant T-K76T), is highly associated with CQ resistance and clinical CQ treatment failure (Djimdé et al. 2001;Wicht et al. 2020).
In Saudi Arabia, CQ resistance was first recorded in 1992 when reports on clinical treatment failures increased (Malik et al. 1997;Alrajhi et al. 1999;Al Arishi et al. 2001). Subsequently, the malaria drug policy in the country was scaled up in 2007 and CQ was replaced with ACT, with artesunate plus SP as a first-line treatment and artemether-lumefantrine as the second-line for the treatment of uncomplicated falciparum malaria (Madkhali et al. 2020;MOH 2018). Since then, some molecular studies have revealed a high prevalence of pfcrt 76 T mutations in isolates from different areas of the country (Al Harthi 2007;Bin Dajem and Al-Qahtani 2010;Bin Dajem et al. 2012). However, those studies only evaluated the mutations at codon 76 of the pfcrt gene, so data on mutations at other codons as well as on pfcrt-related haplotypes are very limited. Interestingly, some previous studies conducted in Africa reported the re-emergence of CQ-sensitive P. falciparum strains several years following the discontinuation of CQ use and the switching of malaria treatment to ACT (Laufer et al. 2006;Dagnogo et al. 2018;Balikagala et al. 2020).
The paucity of data on the status of pfcrt point mutations in Saudi Arabia after 2012 and the potential re-emerging sensitivity to CQ motivated the current study to investigate the frequency and distribution of pfcrt point mutations in P. falciparum isolates in the malaria-endemic Jazan region, 11 years after the ban on the use of CQ to treat uncomplicated falciparum malaria.

Study design and area
The current study was a cross-sectional hospital-based study conducted in Jazan region that targeted febrile patients who were suspected to have malaria. Jazan region is located 16° 17′ North, 42° 43′ East in the southwestern part of Saudi Arabia. The region is bordered by Yemen to the south and by the Red Sea to the west. The region comprises 17 governorates, extends over a total area of 11,671 km 2 and is home to a population of about 1.4 million (GASTAT 2010).
Topologically speaking, Jazan region can be divided into three different areas: the highlands, as represented by the Faifa mountains (which constitute part of the As Sarawat mountain range) at an elevation of over 2000 m above sea level, the foothills at an elevation of 400-600 m and the coastal plains alongside the Red Sea at an altitude of less than 400 m. The aggregated rainfall varies between less than 100 mm/year on the coastal plains and more than 300 mm/year in the highlands (Lashin and Al Arifi 2012). The region also contains some valleys, as well as a number of streams and dams that act as a source of drinking and irrigation water (MEWA 2019). Malaria is endemic in Jazan region, with a few foci of malaria transmission, and transmission peaks between December and March (Madkhali et al. 2020). Anopheles arabiensis is considered the principal malaria vector in the region; however, other species including An. sergentii, An. dthali and An. stephensi have also been recorded (Alahmed et al. 2019;Snow et al. 2013).

Study population
Febrile individuals who presented at selected healthcare facilities in Jazan region and tested positive for malaria between April and December 2018 were invited to participate in the current study, irrespective of their gender, nationality and age. The participants' demographic data (residency, gender, age and nationality) were collected through the use of a semi-structured questionnaire or from the patients' medical records. This was an exploratory descriptive study that did not necessitate the calculation of a power and sample size. However, the required sample size was calculated based on the WHO's guidelines (Lwanga and Lemeshow 1991). Accordingly, at the high prevalence of pfcrt 76 T mutation previously reported, a 95% confidence level and a 5% significance level, the calculation yielded a minimum sample size of 138 P. falciparum isolates. During the study period, a total of 530 participants were screened for malaria, from whom 250 P. falciparum isolates were collected for the molecular examination of point mutations in the pfcrt gene. Subsequently, pfcrt gene were successfully amplified from 235 of those isolates and used for the data analysis.

Blood sampling and examination
A blood sample of approximately 2-3 ml was obtained from each patient and put into an EDTA tube labelled clearly with the participant's identification number, name, gender and age. Promptly thereafter, both thin and thick blood films/ smears were prepared for each blood sample and stained with Giemsa stain in accordance with a standard protocol (WHO 2015). Additionally, dried blood spots were prepared on 3MM Whatman® filter paper and kept in labelled, separate, sealed plastic bag for molecular analysis. The thin and thick stained blood smears were examined microscopically for the presence of Plasmodium parasites and identification of species. From the thick blood film, the parasitaemia (also called parasite density) was estimated by counting the asexual stages of the Plasmodium parasite against 200 white blood cells (WBCs), under the assumption of an average WBC count per μl of blood of 8000 (WHO 2015;Al-Mekhlafi et al. 2021). Some archived malaria-positive slides for the patients examined during the study period were also obtained from the participating healthcare facilities and reexamined for parasite species and parasite density.

Molecular analysis
Extraction of DNA from dried blood spots was done using a Qiagen blood and tissue kit (QIAGEN,DNeasy® Blood & Tissue Kit,Cat. no. 69506,Germany) following the manufacturer's instructions provided in the kit. The extracted DNA was eluted using 100 µl of AE elution buffer, which contained 10 mM Tris-Cl; 0.5 mM EDTA; at pH 9.0 and was kept at − 20 °C until used.

Detection of pfcrt point mutations
The genomic DNA of P. falciparum was subjected to the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique in order to detect mutations at codons 72 to 76 of the pfcrt gene, according to an established protocol (Djimdé et al. 2001). Restriction enzyme (RE) digestion was performed in 20 µl of reaction mixture that included 6-8 µl of PCR product, 1 unit of the specific RE (New England Biolabs Inc., UK) and 1X of the specific buffer provided in the RE kit. The reaction mixture was then incubated for 15-60 min at 37 °C or 50 °C (based on the RE) following the manufacturer's instructions. Then, the mixture was subjected to gel electrophoresis using 4% of TAE-buffered agarose gel stained with Sybr® safe DNA gel stain (Invitrogen, USA). The RE digestion products were visualised under UV light using a Molecular Imager Gel Doc XR System (Bio-Rad, Hercules, CA, USA). The primer sequences, PCR thermal conditions and RE used for the detection of point mutations of the pfcrt gene have been described previously (Atroosh et al. 2016).
The genomic DNA of P. falciparum reference strains that were used by the current study as positive controls were provided by the Malaria Research and Reference Reagents Resources Center (MR4, ATCCW, Manassas, VA, USA). The genomic DNA of P. falciparum strain Dd2 (MRA-150G) was utilised as a positive control for the pfcrt mutated types, while the P. falciparum reference strain 3D7 (MRA-102G) was utilised as a positive control for the wild types.

Statistical data analysis
Statistical data analysis was done using IBM SPSS v20 (IBM Corp., NY, USA). Categorical variables, i.e. the pfcrt point mutations and haplotypes at the studied codons (dependent variable) and other variables such as gender, nationality and age group were described as frequencies and proportions. On the other hand, mean ± standard deviation (SD) or median (interquartile range, IQR) was used to describe quantitative variables, namely, age (years) and parasite density. Associations between the dependent variables and the explanatory variables were examined using the Chi-square test or Fisher's exact test, where appropriate. The significance level of all tests was set at p < 0.05.

Results
A total of 235 P. falciparum-positive patients aged between 2 and 78 years, with a mean age of 30.5 ± 11.6 years, were included in the current study. The majority of the participants (82.1%) were male and 17.9% were female. As regards residency, 69.8% and 30.2% of the participants were from urban and rural areas, respectively. During the study period, malaria-positive samples were collected from all the governorates of Jazan except Altwal, Alraith, Haroob, Fayfa highlands and Farasan Island. The highest percentage (19.1%) of the isolates was from Baysh governorate followed by Jizan (12.8%) and Alharth (11.5%) governorates. About one third (34%) of the participants were Saudi and 66% (155/235) were non-Saudi, and 44.5% (69/155) of the non-Saudi participants were from Yemen. Table 1 displays the general characteristics of the patients who participated in the study. Table 2 shows the frequency distribution of the pfcrt gene point mutations and haplotypes/genotypes in the study sample. Of the 250 isolates subjected to the initial molecular analysis, 235 (94%) were successfully amplified for the pfcrt gene. The pfcrt 76 T CQ-resistant mutant type was found in 66.4% (159/235) of the isolates, while 33.6% of the isolates were found to be carrying the K76 CQ-sensitive wild type. The pfcrt 74I and pfcrt 75E point mutations were each detected in 56.2% of the isolates. On the other hand, isolates were found of all wild types for the pfcrt 73 codon, while only four isolates (1.7%) had pfcrt 72S mutated alleles.

Prevalence and distribution of pfcrt mutations and haplotypes
With regard to the haplotypes/genotypes, four pfcrt 72-76 haplotypes were found to be circulating in the study area. The haplotype identified as having the highest prevalence was the CVIET triple-allele mutated haplotype (56.2%), while the wild haplotype CVMNK was found in 79 isolates (33.6%). In addition, single-allele CVMNT and doubleallele SVMET mutated haplotypes were found in 8.5% and 1.7% of the study isolates. Table 3 shows the associations between the pfcrt point mutations and related haplotypes and the participants' demographic factors and parasitaemia level. In respect of age, a significantly higher percentage of isolates were found to be carrying the mutated pfcrt 76 T allele among patients aged ≥ 30 years (72.4%, 89/123) as compared to patients aged < 30 years (59.8%, 67/112) (χ 2 = 4.128; df = 1; p = 0.042). Also, the percentage of isolates carrying the CVMNK wild haplotype from patients aged < 30 years (40.2%) was significantly higher than the percentage in the isolates from those aged ≥ 30 years (27.6%) (p = 0.042).

Associations of pfcrt mutations with demographic factors and parasitaemia
In regard to parasite density, the percentages of pfcrt 74I and pfcrt 75E mutated alleles were significantly higher (χ 2 = 4.657; df = 1; p = 0.031) in isolates from patients with low parasitaemia (64.1%, 66/103) as compared to those from patients with moderate-to-high levels of parasitaemia (50%, 66/132). Moreover, a significantly higher percentage of the CVIET mutated haplotype (also known as the classical old-world African/Southeast Asian haplotype) was observed in isolates from patients with  low parasitaemia as compared to those from patients with moderate-to-high parasitaemia (64.1%, 50%; p = 0.031).
As for nationality, the pfcrt 76 T allele was observed to be present predominantly among patients from Yemen (94.2%) followed by those from Pakistan (65.5%) and then among Saudi nationals (58.8%) ( Table 3). Interestingly, higher percentages of isolates carrying the mutated pfcrt 76 T allele and CVIET mutated haplotype were found in isolates from non-Saudi patients (70.3% and 57.4%, respectively) as compared to their Saudi counterparts (58.8% and 53.8%, respectively). However, the differences were not statistically significant (p > 0.05). In other words, a higher percentage of the wild-type K76 allele was observed in isolates from Saudi (41.2%) as compared to non-Saudi (29.7%) patients.

Discussion
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 76 T 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 Table 3 Distribution of pfcrt mutant alleles and haplotypes for P. falciparum isolates from Jazan region according to demographic factors and parasitaemia (n = 235)

All values are number (%)
Pfcrt 73 was of wild type (V73) and thus, not included in the analysis Parasitaemia levels: low (< 1,000 parasites/μl of blood); moderate-to-high (≥ 1,000 parasites/μl of blood) Mutant alleles are bold and underlined * Significant association (p < 0.05) † The difference was examined using Fisher's exact test (otherwise, Chi-square test was used)  (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 return 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. It was suggested that removing drug pressure has allowed re-expansion of CQ-sensitive parasites that survived during the period when chloroquine was used (Babiker et al. 2009;Laufer et al. 2010;Mulenga et al. 2021). Moreover, other explanations for the re-emergence of CQ-sensitive parasites include backmutations in the CQ-resistant allele of the pfcrt gene (Fidock et al. 2000) or propagation of a single or a few imported CQsensitive parasites with increased survival fitness over the CQ-resistant parasites (Laufer et al. 2010). Therefore, 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 76 T 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 ( (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 76 T 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. 2011Alareqi 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 76 T 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 2017), 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 pfcrtrelated 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 (Bin 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 76 T mutation and the percentage of the CVMNK wild haplotype were significantly higher in isolates from participants aged ≥ 30 years and < 30 years, respectively. The association between the pfcrt 76 T 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.
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 76 T 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 76 T 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 76 T either between countries or within the same country has previously been reported, and most likely reflects variation in drug pressure between the countries (Kamugisha et al. 2012;Bamaga et al. 2015;Shah et al. 2015;Ndam et al. 2017;Ocan et al. 2019).
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 76 T 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 were 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.

Conclusion
The current study provides important information on the status of CQ resistance in Jazan region, southwestern Saudi Arabia. The findings revealed a potential return of CQ-susceptible P. falciparum strains to the region. While the mutant pfcrt 76 T allele and the triple-mutant haplotype CVIET were detected in 66.4% and 56.2% of the examined isolates, respectively, 33.6% of the isolates were found to be carrying the pfcrt K76 wild allele and the SVMNT wild haplotype. There is a consensus in the research community globally that ceasing the use of CQ in a region could result in the re-emergence of CQ-sensitive P. falciparum strains. This raises the possibility of the reintroduction of this safe and affordable drug, ideally in combination with another antimalarial drug, for falciparum malaria treatment. However, further studies to test this hypothesis are required.
Studies on the molecular markers of antimalarial drug resistance in Saudi Arabia are limited. Therefore, the findings from this study have significant implications for the monitoring of antimalarial drug resistance in the region. Consequently, the continuous molecular surveillance using the pfcrt 76 T point mutation as a reliable marker for CQ resistance as well as further in vitro and ex vivo molecular studies would be highly beneficial. Furthermore, microsatellite loci analyses of larger number of samples set nationally to carefully evaluate pfcrt polymorphisms in Saudi Arabia are strongly recommended.
Health Affairs Directorate of King Fahd Central Hospital, Jazan (Registry no. 086). Before data collection, the patients, and their guardians in case of children, were informed about the objectives of the study and their role. They were also informed that their participation was entirely voluntary and that they could withdraw at any time without giving any reasons whatsoever and without any consequences in respect of their health care. Then, written signed consent was obtained from the adult participants or from the children's parents or guardians. The patients were treated for malaria at the respective healthcare facilities according to the national malaria drug policy.
Consent for publication All authors have agreed and consented to publish the manuscript.

Conflict of interest
The authors declare no competing interests.