The burden of malaria infection has been reduced over the past decades in many countries. Nonetheless, malaria remains an important parasitic disease in humans. To date, there are 5 Plasmodium species able to cause malaria in humans: P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi [1]. Subspecies of P. ovale also infect humans [2]. Human infection is often transmitted through the skin bite with the female Anopheles mosquito while taking a blood meal. Globally, the majority of the malaria cases were attributed to either P. falciparum or P. vivax species. Infections attributed to the other Plasmodium species and/or subspecies are relatively low [3].

Malaria symptoms vary based on the level of host immunity and the pattern of transmission across countries. In the high transmission areas, people develop partial immunity render them capable to tolerate high levels of parasitaemia without symptoms owing to their constant exposure to infection. In contrast, in the low transmission areas, people develop malaria symptoms at low levels of parasitaemia [4]. Malaria diagnosis is usually carried out via traditional diagnostics (Light microscopy and rapid diagnostic tests [RDT]) [5]. Sensitive, quantifiable, high throughput, and field-friendly molecular diagnostic tools have been developed for malaria diagnosis [6].

In Saudi Arabia, malaria is confined to the southwestern parts of the country [7]. Malaria was constrained mostly to foci in Aseer and Jazan regions. In these locations, malaria is mostly imported, and the indigenous malaria is often recognized as low [8]. In areas where the malaria transmission is low, the asymptomatic malaria infections is high and tend to be sub-microscopic. The residual immunity developed in these settings, is believed to be the reason [9]. People with asymptomatic infections may develop symptoms in days or weeks after detection or persist sub-patent for months at unsteady levels of parasitaemia [10]. These asymptomatic sub-microscopic infections constitute an important threat to malaria elimination through local transmission in the low transmission areas.

Jazan is a low transmission malaria district, southwestern Saudi Arabia. Few studies have been performed to describe malaria transmission in the region [11,12]. The current study aimed to provide an update for malaria transmission in Jazan. Our objectives were to identify the infection’s pattern (indigenous, imported, asymptomatic, symptomatic, microscopic and sub-microscopic), to define the causative species and lastly to determine factors that contribute to transmission of malaria in the study’s area. To maximize the sensitivity of malaria diagnosis, a species-specific nested polymerase chain reaction (nested PCR) was used in addition to the conventional diagnostics (microscopic examination and the RDTs). Taking the nested PCR results as a reference, the diagnostic performance of the microscopy and the RDT were also investigated in our study.

Prompt diagnosis of malaria in the low transmission settings is important for treatment and infection control. Molecular diagnostic tool capable to detect challenges that arise with the low transmission, is crucial for countries poised to achieve malaria elimination, like Saudi Arabia. In this survey we included a species-specific nested PCR tool into the traditional malaria diagnostics to provide useful insights on several aspects of malaria transmission in Jizan district.

In the current study, the point prevalence of malaria in the study’s population was ~11% with the nested PCR. The vast majority of cases (83.3%; 25/30) was designated as imported malaria, coinciding with earlier reports. El Hassan and his co-workers have reported high number of imported malaria cases in Jazan [11]. In Hail, another Saudi district, imported malaria has been identified in 25% of patients based on microscopic examination, 2014–2015 [16]. Notably, the international borders between countries represent a big challenge for countries seeking malaria elimination, as stated elsewhere [17]. In agreement with this observation, the majority of imported malaria cases reported in our study (44%; 11/25) were originated from Yemen. The Republic Yemen has been frequently considered an important source of imported malaria infections into Saudi Arabia. About 32% of all imported infections detected in all Saudi area have been arised from Yemen, according to Al Zahrani et al. [18] 2015–2017. The imported malaria sustains potential for indigenous malaria transmission, new Plasmodium species development and emergence of drug resistance [19]. Thus, imported malaria represents a serious threat to infection elimination. In The current study, 16.6% (5/30) of the detected malaria cases were indigenous. Significant reduction in the number of the locally acquired malaria cases has been exhibited in earlier studies in Jazan and elsewhere in Saudi Arabia. Researchers attributed the reduction of the native malaria cases in the study site to the effective control protocol and to the low rainfall practiced, 2000–2014 [11]. In Taif, a low transmission area, western Saudi Arabia and close to Mecca, much lower estimates of both imported and indigenous malaria have been reported in a hospital-based population [20]. Perhaps, the endemicity pattern of malaria in the study’s site, the study’s population, the clinical samples and the diagnostic methods used could explain the variation of the imported and indigenous malaria burden among studies.

In the current study, malaria cases were detected throughout the year. Nonetheless, most of cases were identified in February–March and September–October peak periods. This finding might be ascribed to increase travel at these periods. In Aseer, an area near Jazan, the malaria transmission has peaks in February and April following the rains [21]. The local climate and its effect on human-vector activities may have a role for determination of months potentials for peak malaria transmission. Adults aged 15–24 years and males were identified as high-risk groups in the study site, consistent with previous reports [21,22]. In our study’s location, adult males are more prone to mosquito bite than females and children because they are more likely to be outdoors. We can also point to the fact that most of the migrants who crossed the borders to Jazan, during the study duration, were adult males coming from malaria-endemic areas. In contrast, Jenkins and her co-workers have reported malaria more in females than males in malaria endemic area of Kisumu County, Kenya [23]. This discrepancy may be related to the customs and the traditions of the study’s site. In Jazan, by virtue of the customs and the traditions, females wear clothes that cover the entire body and protect them from mosquito bites.

The Plasmodium species distribution in our study was 76.6% P. falciparum, 16.6% P. vivax and 6.6% mixed falciparum/vivax infections. The dominance of P. falciparum over P. vivax infections was evident in our study, consistent with one report [24] and inconsistent with another [16]. Hasona et al. [16] have reported malaria cases attributed more to P. vivax than to P. falciparum based on RDT. Perhaps, the discrepancy in species distribution between the 2 surveys might be related to the country of the origin of migrants and the Plasmodium species endemicity situation. In our study, the imported malaria cases were primarily originated from P. falciparum-endemic countries (Yemen, India, and Sudan) while in Hasona et al. [16], the imported malaria cases have been originated mainly from P. vivax-endemic malaria countries (Afghanistan, Pakistan and Iraq). Also, in our study, the nested PCR revealed a low proportion (0.7%) of mixed falciparum/vivax infections in the study location. Bin Dajem, in year 2015, has detected a lower proportion (1.9%) of mixed falciparum/vivax in the study’s site [25]. Gupta et al. [26] have reported a much higher proportion (45.5%) in India. Mixed malaria cases have been overlooked in several Saudi studies, Dawoud et al. [27]’s study is one of these studies. These variations might be due to different methodology adopted in these studies. Mixed malaria infections are often overlooked in studies relying on the classical diagnostic tools. No malaria case attributed to either P. ovale or P. malariae was detected in the study’s population, concordant to Dafalla et al. [12].

Approximately 73% of the detected malaria cases, in our study, were symptomatic (i.e., presence of fever) and −27% were sub-patent (i.e., absence of fever). Detection of the sub-patent malaria infection is important for the interruption of malaria transmission. The relevance of the symptomless infection rests on the fact that it can transmit malaria in the study’s setting [28]. The malaria parasites present in these cases at densities below the limit for microscopic detection and therefore, they were missed detection by the light microscopy. These sub-patent malaria infections were found frequently attributed to P. vivax as sole infection species in three cases and concomitant with P. falciparum (mixed infection) in one case. Interestingly, out of these eight asymptomatic cases, seven were missed by microscopy (sub-microscopic). The sub-microscopic asymptomatic malaria infections have been demonstrated a contribution to malaria transmission in Jazan [29] and elsewhere in Thailand [30]. These sub-patent infections were most prevalent in patient above 15 years, coinciding with previous report [31]. According to one study, populations with higher levels of immunity (i.e. aged >15 years) would maintain the malaria parasites at lower densities enough to be easily missed detection by the microscopy [32].

In this study, we also examined the performance of the three malaria diagnostics. Both the microscopy and the RDT proved less sensitive than the nested PCR, as expected, and already described in a previous study [33]. Microscopy showed the low sensitivity for diagnosis of sub-patent malaria. Seven asymptomatic malaria cases were not recognized by microscopy. Interestingly, four of these sub-microscopic cases were identified by the immunochromatographic test. These four malaria cases might be for patients given anti-malarial treatment and having malaria parasite surface antigens in their blood. The persistent of parasite histidine-rich protein 2 antigen in patients given antimalarial treatment has been previously demonstrated [34]. Consistent with what has been described in the literature [35], the Paramax-3^TM RDT detected malaria infection in 14 patients, not confirmed by the nested PCR. Plasmodium species deficient in the hrp2 gene (e.g. due to deletion) are a possible source of false-negative HRP2 RDTs, according to one study [36]. Of concern, the RDT-false positive results lead to non-essential prescription of the antimalarial drugs that carries side effect on health and may contribute to the emergence of resistant strains [37]. Performance variations of different RDTs have been described based on the transmission settings. Ranadive et al. [38] have attributed the low sensitivity of the RDTs in a hypoendemic malaria setting of Swaziland, to the high proportion of low-density infection among symptomatic patients. On contrast, Kurup and Marks, in a study carried out in Guyana, a high transmission setting, have reported a sensitivity to Paramax-3^TM comparable to that of the microscopic examination [39].

The current study was not free of limitations. Despite of the important data reported in our survey, the survey’s data could not apply for the whole district, being a hospital-based. Moreover, our survey’s data might be biased by the non-representation of certain population groups in the study’s population such as pregnant women, children under 5 years and patients with severe complicated malaria. Also, in the current study, the levels of parasitaemia were not estimated in malaria-positive cases and in our evaluation of the microscopic examination and the RDT, we primarily relied on an assumption taken with the superiority of the nested PCR to the other two tests. Detection of the degree of parasitaemia were not given a consideration our study as the evaluation of the conventional diagnostic tools was not a core purpose of our study. Lastly, the small number of the PCR-confirmed malaria cases made the analysis of some collected data very challenging [40].

To conclude, the burden of malaria in Jazan district is relatively high. The largest portion of malaria was imported, largely from Yemen. Infections were attributed mainly to P. falciparum and to a lesser extent to P. vivax. Despite its relatively low number, falciparum/vivax mixed infections were detected in the study’s site. Considerable proportion of asymptomatic sub-microscopic infection was also described. Infections were frequent in adults, males, in patients presented with fever and in patients with recent travel to malaria-endemic areas. Lastly, the low sensitivity of microscopy and the Paramax-3^TM were evident in comparison to the nested PCR. We suggest incorporation of a molecular tool into the traditional malaria diagnosis in Jazan to overcome challenges arising from the low transmission of malaria in the district.