Background
Following global decline in malaria over past decades [
1], the importance of accurately describing transmission in (pre-) elimination areas is widely documented [
2]. Use of data collected at health facilities might not be robust in some areas as data are highly dependent on health-seeking behaviour and the effectiveness of the health system [
3]. Evaluating transmission intensity by determining exposure to malaria-infected mosquitoes is challenging when mosquito numbers are low [
4]. Serological evidence of exposure to malaria, by the presence of anti-malarial antibodies, offers a measure of past infection and can determine temporal [
5] as well as spatial trends in transmission [
6]. In low-endemic settings, long-lasting antibody responses may be easier to detect than parasite carriage in the human population or infected mosquitoes. Moreover, the absence of anti-malarial antibodies in certain age groups has been used as evidence of the cessation of transmission in Greece and Mauritius [
7,
8]. The presence of serological evidence of exposure to malaria alongside molecular testing to detect parasites are currently considered to be most appropriate metrics in areas of low transmission and under elimination [
3,
9].
In Iran, a national strategic plan for the elimination of malaria was approved by High Council for Health and Food Security in 2010 to stop local transmission by 2025. In this regard, Iran achieved a substantial decline in malaria incidence according to WHO Malaria Reports [
1,
10]. The burden of malaria decreased gradually from nearly 97,000 cases in 1991 to 16,000 in 2007 [Center for Diseases Management and Control (CDMC), Tehran, Iran, unpublished data]. A further reduction was reported in 2014 with 1251 being the total number of cases (75 % decrease since 1991). Iran is considered to be in the elimination phase for malaria since 2009 [
11]. Areas of malaria transmission are found in the southern part of the country, which accounts for more than 90 % of the total number of cases [
12]. The majority of cases, 70 % in 2014 (CDMC, Tehran, Iran, unpublished data), is due to imported cases from neighbouring Pakistan and Afghanistan [
13].
Investigating the application of different metrics in order to demonstrate the absence of transmission and simultaneously show the likelihood of success and outcomes of malaria interventions during pre-elimination and elimination programmes is essential in Iran. Previous serological studies in Bashagard and Ghale-Ganj Districts, southern Iran, found seroprevalence to
Plasmodium vivax and
Plasmodium falciparum to be 1 % or less [
12,
14]. No parasite carrier was detected by microscopy and/or molecular testing. Although seroprevalence was low in these districts, higher transmission patterns in neighbouring Sistan and Baluchistan Province are to be expected, based on previous incidence data [
1]. This study was designed to determine serological and parasitological transmission levels of
P. vivax and
P. falciparum malaria in Chabahar District, Sistan and Baluchistan Province in the malaria elimination phase in southeastern Iran.
Discussion
Iran is certified as an elimination area since 2009 [
11] and only approximately 1250 clinical cases were reported in 2014, of which the majority originated in the southeastern part of the country (CDMC, Tehran, Iran, unpublished data). As transmission reaches eliminating levels, identifying areas with remaining transmission or absent transmission requires new approaches. In this study, transmission levels of
P. falciparum and
P. vivax malaria were determined in Chabahar city and surrounding villages, Sistan and Baluchistan Province in the malaria elimination phase in southeastern Iran. There was no parasitological or serological evidence of recent local transmission, indicated by both an absence of microscopic or sub-microscopic (nPCR) infections, and weak or absent serological responses to either species in children up to the age of 5 years.
None of the children up to the age of 5 years in the villages showed seropositivity to
P. falciparum, and only four were found in Chabahar city (2 %). Their adjusted antibody levels as represented by OD values were invariably low and close to the cut-off level, suggesting that they may not be related to recent infection or, given the age group, perhaps any infection. A number of alternative approaches are available to determine positivity thresholds in antibody tests [
22] and indeterminate ranges are a common feature in commercially available serological assays [
23]. When more conservative threshold of five standard deviations plus the mean of the lower Gaussian distribution was used none of the young children in the city was classified as seropositive. This suggests antibody responses in this age group were low and serological evidence of exposure to
P. falciparum malaria is weak. Regarding
P. vivax, using the more conservative threshold for positivity, only six out of the 15 seropositive 1–5 years old children remained positive. Similarly, for adults, serological responses were considered to be weak as only a minority mounted multi-antigenic responses. Approximately 15 % of the seropositives in the city were positive for more than one antigen and 25 % in the villages. Recent studies have likewise reported no parasitological evidence of transmission and low seroprevalence in the neighbouring malaria-endemic provinces of Hormozgan (Bashagard) and Kerman (Ghale-Ganj) [
12,
14]. The detected lower overall seroprevalence in these studies is most likely due to lower levels of historical exposure. It is clear that for broader use of serological approaches in studies, regions and countries a standardized assay would be needed, such as those available in standardized anti-malarial antibody detection assays for the screening of blood products [
24]). Nevertheless, the presented data from parasitological and serological tests strongly support low or absent levels of recent malaria transmission in these settings in Chabahar District.
The best model for age of
P. falciparum seroconversion in Chabahar city was one with two forces of infection changing 21 years before sampling was done (
P = 0.018). Seroprevalence in the city for individuals aged under the age of 21 was ten-fold lower compared to those aged 21 years and older. The timing of this change, 1991, coincides with a previously described dramatic drop in malaria cases in the early 90 s, both nationwide [
25] and in the southwestern part of the country [
13]. An alternative, but not mutually exclusive explanation, is that this could reflect increased risk of exposure in older individuals (>21 years), for example due to more frequent travel to malaria-endemic areas in neighbouring countries (e.g., Afghanistan and Pakistan). In the villages, a change point was seen at 4 years before sampling was done (2008;
P = 0.039) suggesting transmission has only declined recently in these areas.
This might be explained by the up-scaling of different interventions during elimination strategies (CDMC, Tehran, Iran, unpublished data), such as active case detection, case management, early diagnosis, prompt and effective treatment [artemisinin combination therapy (ACT) plus single dose of primaquine on day 3 as the first-line recommended therapy for
P. falciparum uncomplicated malaria and chloroquine with 8 weeks primaquine for radical treatment of
P. vivax], indoor residual spraying, the distribution of LLINs, larviciding, and improved diagnostic capacities in health facilities (microscopy and rapid diagnostic testing) [
26]. In addition, Iran is working to improve its cross-border collaborations and to apply a malaria early warning system and outbreak preparedness plan for epidemics by increasing training of microscopists, rural malaria mobile teams, and community volunteers to overcome this problem [
27].
Moreover, no change in SCR was seen for
P. vivax, with values similar in both rural and urban settings. This may be due to ongoing exposure to infection with this parasite or boosting of antibody levels by hypnozoite-derived infections. However, it may also be due to small sample size in addition to low seroprevalence [
28]. This requires further investigation.
The overall
P. falciparum and
P. vivax seroprevalence presented here (13 and 10 %, respectively) was lower than previously described levels in other countries in the Eastern Mediterranean region, such as Somalia (18 and 19 %) [
6], Djibouti (32 and 3 %, adults only) [
29], and Yemen (32 and 3 %, children only) [
30]. These studies took place between 2002 and 2011, while the data presented here are from 2012. The differences in seroprevalence are likely to be related to the lower level of transmission in Iran in recent years. Increasing collection of serological data in the region, alongside other metrics of malaria, has the potential to help in further characterizing regional transmission patterns.
As no parasite infections were detected with either microscopic or nPCR methods, risk factors to describe exposure to malaria could only be examined using serological outcomes. The increased likelihood of seropositivity to
P. vivax remained twice as high for villagers in comparison with city dwellers in the adjusted model. It seems that ‘previous’
P. vivax transmission was higher in the villages surrounding Chabahar city than in the city itself, indicating higher potential receptivity for the re-introduction of malaria in the villages in this district. More geospatially explicit work would allow specific targeting of these areas to make their use more cost-efficient [
31]. Iran has several intervention policies in place, such as the distribution of bed nets free of charge since 2005 [
10], vector control strategies (such as microbial larviciding), as well as active case detection, case management, artemisinin-based combination therapy as the first-line recommended therapy for
P. falciparum uncomplicated malaria, and improved diagnostic capacities. These could help maintain the absence or prevent the re-introduction of malaria transmission in the study areas with very active human population movements between Iran, Afghanistan and Pakistan, common parasite-vector fauna, as well as similar economic-cultural inhabitants, which provides an environment that is in favour of parasite transmission. Further monitoring of seroprevalence in residents of the study areas is considered highly useful given the low levels of transmission and population movement between malaria-endemic areas of Pakistan and Afghanistan that might affect transmission in these settings.
Interestingly, SES influenced the trend for seropositivity to
P. falciparum, but not
P. vivax. This observation might be due to
P. vivax antibody levels being predominantly associated with relapses rather than new infections, as recently described in Papua New Guinea [
32], and thus not influenced by household characteristics. The shown similarity in
P. vivax (but not
P. falciparum) seroconversion curves between city and village settings as well as the fact that there was weak to absent parasitological or serological evidence of recent
P. vivax transmission in children, support this hypothesis. However, the latter may also be due to a higher risk of
P. vivax in older individuals (e.g. due to work). It would be useful to confirm these findings in other
P. vivax endemic, but eliminating, areas. These assumptions would be strengthened by inclusion of a broader range of antigens, ideally including those related with hypnozoite carriage (as yet unidentified).
Authors’ contributions
SZ and AR designed the study and supervised sample collection. SZ, AAM, FK and LH carried out serological assays. LH, SZ and CD analysed the data and wrote the manuscript. All authors read and approved the final manuscript.