J Arthropod-Borne Dis, June 2017, 11(2): 172–181 Gh Hassanpour et al.: Asymptomatic Malaria … 172 http://jad.tums.ac.ir Published Online: May 27, 2017 Review Article Asymptomatic Malaria and its Challenges in the Malaria Elimination Program in Iran: a Systematic Review Gholmreza Hassanpour 1,2, Mehdi Mohebali 1,2, Hojjat Zeraati 3, Ahmad Raeisi 1, *Hossein Keshavarz 1,2 1Center for Research of Endemic Parasites of Iran, Tehran University of Medical Sciences, Tehran, Iran 2Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medi- cal Sciences, Tehran, Iran 3Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 30 Apr 2016; accepted 21 May 2016) Abstract Background: The objective of this study was to find an appropriate approach to asymptomatic malaria in elimina- tion setting through a systematic review. Methods: A broad search was conducted to find articles with the words ‘malaria’ in their titles and ‘asymptomatic’ or ‘submicroscopic’ in their texts, irrespective of the type of study conducted. The Cochrane, Medline/Pub Med, and Scopus databases, as well as Google Scholar were systematically searched for English articles and reports and Iran’s databases- Iran Medex, SID and Magiran were searched for Persian reports and articles, with no time limitation. The study was qualitatively summarized if it contained precise information on the role of asymptomatic malaria in the elimination phase. Results: Six articles were selected from the initial 2645 articles. The results all re-emphasize the significance of asymptomatic malaria in the elimination phase, and emphasize the significance of diagnostic tests of higher sensitivi- ty to locate these patients and perform interventions to reduce the asymptomatic parasitic reservoirs particularly in regions of low transmission. However, we may infer from the results that the current evidence cannot yet specify an accurate strategy on the role of asymptomatic malaria in the elimination phase. Conclusion: To eliminate malaria, alongside vector control, and treatment of symptomatic and asymptomatic pa- tients, active and inactive methods of case detection need to be employed. The precise monitoring of asymptomatic individuals and submicroscopic cases of malaria through molecular assays and valid serological methods, especially in regions where seasonal and low transmission exists can be very helpful at this phase. Keywords: Malaria, Asymptomatic infection, Elimination Introduction Across the globe, 3.3 billion people are at risk of malaria. In 2013, 198 million people were affected with the disease, of which 584000 people died (World Health Organi- zation 2014). A 50% reduction in the num- ber of malaria cases by 2015 was announced as a Millennium Developmental Goal. Current- ly, 99 countries are malaria-free and 34 coun- tries are in the phase of elimination (Gueye et al. 2013). The countries that are in the elim- ination phase have put different strategies on their agendas, such as general screening and focusing on specific foci (World Health Or- ganization 2013). In such countries, at the same time that transmission reduces, the major share of transmission will take place through collective and focal transmission (Ministry of Health and Mdical Education 2012). Probably, the infected asymptomatic indi- vidual can become a source of parasitic trans- mission to healthy individuals under favora- ble setting, considered a serious challenge to *Corresponding author: Dr Hossein Keshavarz, E-mail: hkeshavarz@tums.ac.ir http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 172–181 Gh Hassanpour et al.: Asymptomatic Malaria … 173 http://jad.tums.ac.ir Published Online: May 27, 2017 malaria control and elimination worldwide (Lin et al. 2014). The popular belief is that in low transmission settings the proportion of asymptomatic individuals is less than that in areas of greater transmission severity. How- ever, community-based studies have shown that, although an increased transmission state is associated with an increase in the reservoir share, but even in low transmission areas the asymptomatic cases make up for 60% of the infected population (Sturrock et al. 2013). Therefore, it seems that in low transmission settings the malaria infection is very likely to be asymptomatic (WHO 2013). While symp- tomatic cases are removed from the disease reservoir faster because of treatment. Under such circumstances, communities appear to need interventions that address beyond symp- toms (Sturrock et al. 2013). Under circumstances in which many suc- cessful and unsuccessful attempts at malaria elimination have been made worldwide, this review was designed to find an appropriate strategy for the detection and elimination of asymptomatic malaria. The summarization of these experiences in the form of a systematic review can effectively help clarify these experi- ences and in turn help national policymakers who are trying to eliminate malaria in Iran Materials and Methods Study design This study has systematically searched all the studies, reports and documentations relat- ed to malaria and the role of asymptomatic malaria in the elimination phase -with no time limitation- up to July 2015. Inclusion criteria In this systematic review, all the articles that had questioned or responded to the main ques- tion of the study, i.e. ‘the role and significance of asymptomatic malaria cases in the pre-elim- ination and elimination phases of malaria’ irre- spective of the study design- were selected. Search strategy The Cochrane, Medline/PubMed, and Sco- pus databases, as well as Google Scholar were systematically searched for English articles and reports and Iran’s databases -IranMedex, SID and Magiran were searched for Persian reports and articles, with no time limitation. We performed a search of high sensitivity to find the relevant articles. In short, “(Malaria in Title) AND (asymptomatic OR sub micro- scopic OR sub-microscopic OR low para- site) were searched in various databases. Selecting studies, quality assessment and data extraction all the article titles were first entered into reference manager software (End- Note). After omitting the duplicate cases, they were prepared for the initial screening. In the first stage, the article titles were reviewed and those articles that were obviously irrelevant to the study objective were removed from the primary databank. In the next stage, two persons studied the titles and abstracts and the articles that were selected by both per- sons were finalized. If only one person chose an article, then that article was discussed in a meeting and the decision to include or ex- clude it in the study was made thereafter. At this stage, the selected articles’ full texts were obtained and those articles that had specifi- cally answered the following two questions were selected as candidates for inclusion. The two questions were, “Has the article discussed the topic of Asymptomatic and/or Submicro- scopic malaria? Have these topics been ap- plicable in the elimination phase of malar- ia?” If the answers to these questions were ‘yes’ then the article would be included in the study. The selected studies were thorough- ly reviewed by two persons and their opin- ions were shared with each other in a joint meeting. If the overall impression of the ap- plicability of the study results in malaria elim- ination was the same then that article has been selected. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 172–181 Gh Hassanpour et al.: Asymptomatic Malaria … 174 http://jad.tums.ac.ir Published Online: May 27, 2017 Results The systematic review yielded 2645 arti- cles in all (Medline/PubMed: 718, Magiran: 210, IranMedex: 226, SID: 74, Web of Sci- ence: 668, Scopus: 748, other databases such as EMB Reviews and Cochrane: 1). After omitting the duplicate cases 1322 articles were sorted for title review. Upon reviewing the article titles, 143 articles, and upon review- ing the abstracts 61 articles remained in the databank, respectively. The full texts of these 61 articles were obtained and eventually, upon studying those, only six articles were final- ized (Fig. 1, Table 1). The most important reason for exclusion in the final stage was lack of a direct relation and an applicable re- sult in malaria elimination settings. In summary, the selected articles empha- sized the significance of the role of asymp- tomatic malaria in the elimination phase. They also emphasized the need for diagnostic tests of higher sensitivity to detect these cases and the performance of interventions aimed at re- ducing parasite reservoirs -with an emphasis on asymptomatic cases. Lind blade et al. published an article on the significance of asymptomatic malaria in 2013. They stated that although malaria con- trol has had many positive effects in the world, there is not much certainty regarding the current interventions aimed at eliminat- ing malaria in many endemic countries. In this review, a great share of the infected cases of malaria has been attributed to asympto- matic individuals who were not seeking treat- ment. These circumstances become even more challenging in regions where Plasmodium vivax contributes more to the burden of disease be- cause of its dormant liver stage. They con- clude by stating the important role asympto- matic malaria has to play in its transmission and that in order to eliminate the disease, in- terventions must target the reduction of par- asite reservoirs in both low and high trans- mission setting (Sturrock et al. 2013). “Targeting asymptomatic malaria infections: active surveillance in control and elimination” -another study published in 2013 by Sturrock et al. underscores that scale up malaria control programs in various countries have reduced the focal transmission of disease. In a small- er scale, each focus consists of hotspots such as households and groups of households with higher malaria transmission and con- sistent parasite environments throughout the year. This infection is clustered in specific populations (hotspots) that have high-risk de- mographic characteristics. The authors believe that the identification of these groups -both geographically and demographically- is an important strategy toward reducing local parasite reservoirs and disrupting the trans- mission chain in low transmission or elimi- nation setting (Ministry of Health and Med- ical Education 2012) The current approaches toward malaria elimination are based on the recommenda- tions of the Global Malaria Eradication Pro- gram of 1960. However, many countries face multiple challenges in the fourth phase of elim- ination, such as, the imported cases of infec- tion, which apparently requires a regional ap- proach. To eliminate malaria, focus needs to be laid on the detection and elimination of infec- tious foci through active and inactive methods of patient detection. In this approach, along- side vector control, both symptomatic and asymptomatic patients should be treated cor- rectly. Since imported cases can be a source of disease transmission, malaria elimination programs should have precise plans for these cases as well (Moonen et al. 2010). In countries that have gone through the control phase, to ensure malaria elimination and monitor transmission reduction, diagnos- tic tools applicable in the field are necessary to detect asymptomatic and low-density par- asitic cases for extensive screening and treat- ment (Lo et al. 2015). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 172–181 Gh Hassanpour et al.: Asymptomatic Malaria … 175 http://jad.tums.ac.ir Published Online: May 27, 2017 In an article Lin et al. (2014) emphasized that malaria elimination can be achieved by eliminating the infected human reservoirs (in- cluding asymptomatic cases). A better under- standing of the role of submicroscopic cases as a source of transmission can greatly help to adopt an active surveillance approach. Ac- cording to this article, the results of screen- ing have shown the capability of RDTs in detecting asymptomatic cases at a threshold of 200 parasites per microliter, which is a submicroscopic threshold. Therefore, it seems that in order to eliminate, we need newer methods of higher sensitivity to be able to detect potential transmission agents at any scale (World Health Organization 2013). Molecular diagnostic tests have shown that even in low endemic regions, asympto- matic malaria reservoirs are more than they were believed to be. Individuals that can be diagnosed with microscopic or submicroscop- ic methods have submicroscopic gametocyte density. Bousema et al. (2014) believed that, in order to eliminate malaria, interventions should target both symptomatic and asymp- tomatic infections. In regions where there is seasonal and low transmission of malaria, even a small percentage of infected persons are sufficient to re-start malaria transmission. De- pending on various conditions, the transmis- sion of infection from mosquito to human is different, however, as a barrier to malaria elim- ination, asymptomatic and submicroscopic cas- es should be monitored through appropriate molecular methods (Lindblade et al. 2013). Fig. 1. Flow chart demonstrating database searches, identification, screening and selection of included studies http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 172–181 Gh Hassanpour et al.: Asymptomatic Malaria … 176 http://jad.tums.ac.ir Published Online: May 27, 2017 Table 1. Characteristics of included studies Challenges/ acceptability Recommended policy Aim Type of study Year Author Can molecular methods em- ployed to detect asymptomatic individuals help reduce trans- mission? Interventions should target both symptomatic and asymptomatic individuals, depending on the circumstances Asymptomatic malar- ia and public health relevance Opinion 2014 Bousema et al. Lack of a unified definition of asymptomatic malaria By choosing efficient interven- tions for asymptomatic patients, parasitic reservoirs should be destroyed. The role of asympto- matic malaria in ma- laria transmission and interventions to achieve malaria elim- ination Review 2013 Lindblade et al. Absence of scientific evidence supporting or opposing the role of submicroscopic malaria in transmission Advising molecular diagnostic tests in longitudinal studies and exploring the role of reservoirs in malaria transmission The role of submicro- scopic parasitemia in malaria transmission Review 2014 Lin et al. There is no evidence of the cost-effectiveness of these in- terventions in the long run RACD appropriate to low transmission settings and PACD appropriate to low/medium transmission Potential role of ACD in malaria control and elimination Policy forums 2013 Sturrock et al. Difficulties are observed in countries with little resources; also the companies that can tend to these priorities are small companies that need support The need for diagnostic tests with little parasitic density in asymptomatic persons To propose a research agenda for the tools required for malaria eradication Review 2011 Malera consultive group There is no the global cam- paign, national elimination cannot succeed with continued importation. Establishment of a surveillance system connected to rapid re- sponse surveillance over time To review the activi- ties needed to achieve and maintain malaria elimination Opinion 2010 Moonen et al. Discussion We found only a few studies with goals completely relevant to our research question. None of the studies had discussed with cer- tainty the role of asymptomatic malaria and the correct approach toward these cases, par- ticularly in malaria elimination programs. The evidence suggested that asymptomatic ma- laria probably has a major role to play in ma- laria transmission. Communities need tools of higher sensitivity to detect asymptomatic and low-parasitic density cases. Malaria elim- ination requires interventions aimed at reduc- ing parasite reservoirs, both in high- and low- transmission setting. In this systematic review, we tried to re- trieve all the relevant data. However, most of the selected articles including review articles were expert commentaries and recommenda- tions. However, the scientific community can- not cite such evidence with great certainty when it comes to the evidence pyramid. The lack of interventions and prospective cohort studies relevant to the research question on one hand, and the high significance of cli- mate, parasitic species and vector differences on the other, does not allow a conclusion to be reached with certainty on the strategy of dealing with asymptomatic patients -particu- larly at the elimination phase of the disease. Although experts have emphasized the role of asymptomatic carrier in various studies, but the interesting point is the absence of a unified definition of asymptomatic malaria (Moonen et al. 2010, Hassanpour et al. 2011). Most studies have described asymptomatic malaria by the presence of sexual or asexual multiplication parasites and/or absence of acute clinical symptoms (usually fever) during a spe- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 172–181 Gh Hassanpour et al.: Asymptomatic Malaria … 177 http://jad.tums.ac.ir Published Online: May 27, 2017 cific period. Some studies have focused on the parasite density threshold, and have con- sidered febrile patients with higher than pre- determined threshold parasite densities symp- tomatic. Various studies have set different criteria and given different definitions for be- ing symptomatic, such as, different follow- up periods for becoming symptomatic, tak- ing into account one symptom (mostly fever) and/or all clinical symptoms (Sturrock et al. 2013). Perhaps the first shortcoming in adopt- ing an operational approach toward asympto- matic malaria is the lack of a unified defini- tion of asymptomatic malaria in different ref- erences. Asymptomatic malaria can become symp- tomatic due to relative immunity, frequency of exposure to malaria infection, increased age (independent of repeated exposure), and cross-protection resulting from relative im- munity from exposure to multi clonal infec- tions. Among various factors, age and previ- ous exposure are the most important deter- minant factors of immunity in individuals (Sturrock et al. 2013). Moreover, sometimes, symptomatic per- sons who have not been treated enter the chronic phase of the disease and reach a par- asite level lower than threshold -which is not observable under the microscope. Another important group are those persons who have recently received treatment or have been di- agnosed early (before becoming symptomat- ic) following a diagnostic test such as PCR, and/or have a parasite density level that has never become observable under the micro- scope (Lindblade et al. 2013). Hence, we may conclude that asymptomatic malaria in- cludes many different types of patients. In addition to these groups, we must keep in mind that some asymptomatic individuals are patients who have received treatment in the past, those who have probably appeared asymptomatic because of incomplete treat- ment or relative drug resistance. This matter requires meticulous studies to identify the genotype of the parasite, in terms of whether it is a residual infection or a new one. Nev- ertheless, in addition to the aforementioned complexities, associated infections with par- asites such as Ascaris lumbricoides and Schis- tosomia hematobium too can complicate the detection of these individuals because of cross reactivity (Sturrock et al. 2013). Evidence suggests that all species of ma- laria can result in asymptomatic disease. How- ever, since P. falciparum and P. vivax are more prevalent, most asymptomatic patients are infected with these two Plasmodium spe- cies. The P. vivax species create immunity more rapidly as compared to falciparum spe- cies, hence raising the possibility of control- ling the parasite density as well. Although there are fewer P. vivax studies than there are P. falciparum ones, but it seems that in this species too, an increase in the number of gametes raises the possibility of the insect’s infectivity. Various studies in different set- tings and with different diagnostic tools have shown entirely variable results, such that, we cannot draw an association between the asymp- tomatic cases of malaria and its different spe- cies (Sturrock et al. 2013). Another challenge in approaching asymp- tomatic malaria is the existent knowledge of the true role of the affected individuals in disease transmission. The uncertainty sur- rounding the role of submicroscopic malaria in disease transmission can be dangerous from two aspects. On one hand, asymptomatic malaria is considered a serious challenge in malaria control and elimination in many parts of the world, as it can under favorable condi- tions act as a reservoir and cause parasite transmission. On the other hand, if these cases are not treated effectively then they can provoke the global drug resistance prob- lem (World Health Organzation 2013, Lin et al. 2014). Many believe that some of these individuals eventually become symptomatic. However, there is no certainty as to whether these individuals have been affected with a http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 172–181 Gh Hassanpour et al.: Asymptomatic Malaria … 178 http://jad.tums.ac.ir Published Online: May 27, 2017 new febrile illness or that re-infection has taken place (Sturrock et al. 2013). In addition to all the aforementioned di- agnostic issues and reasons of presentation of these cases, the important question is the Infectivity of asymptomatic individuals. The general belief is that gametocyte density is the most important determinant factor of in- fection transmission from an infected individ- ual to an anopheles vector (Diagnoses. 2011). So, the lower the gametocyte carriage den- sity, the less significant the issue of infectiv- ity. Theoretically speaking, the possibility of transmission exists with even a single game- tocyte per microliter. However, the current evidence does not point to a robust relation- ship between gametocyte density and asymp- tomatic individuals’ infectivity. Presently, it may be said that the gametocyte is an incom- plete surrogate for transmission. The evi- dence on gametocyte carriage among asymp- tomatic cases is also insufficient. Direct skin test feeding, observance of oocysts in the Anopheles midgut, and membrane feeding apparatus studies may to some extent answer these questions. In addition to gametocyte density, other factors such as the vector and host also affect this transmission (Sturrock et al. 2013, World Health Organzation 2013). Under the most favorable conditions and in standard laboratories, Giemsa-stained blood film optical microscopy can detect more than 10 parasites per microliter, while, ordinarily, approximately 100 parasites per microliter should be present in 5 microliters of blood. Methods such as qPCR have the ability to detect almost double the microscopic meth- od, considering the groups at risk and the community in which the examination is being done (Lindblade et al. 2013). Depending on the diagnostic method, be it microscopy, RDT or PCR, the detection of asymptomatic cases has yielded completely different results in different communities ( Haghi et al. 2012, Sturrock et al. 2013, Bousema et al. 2014). The most important applied difference be- tween control and elimination programs is the focus on detection of and intervention on symp- tomatic and asymptomatic infections (Moonen et al. 2010). As the prevalence of the disease falls, activities should focus on subclinical and low-density infections and reservoirs present in specific geographic and demographic areas. No doubt, to achieve the goals of disease elimination and eradication, we must be able to detect malaria infection with applicable diagnostic tests, something that is not possi- ble with the current microscopic methods, where PCR is an expensive technique and not practical for the field (Laishram et al. 2012). In low transmission or malaria elimination conditions, transmission takes place focally. Therefore, the approach of targeted detection of these infection clusters becomes extreme- ly important for reducing local reservoirs and disrupting the transmission chain (Min- istry of Health and Education 2012). Sub- microscopic infections in high transmission regions (where parasite prevalence in the com- munity exceeds 75% microscopically) are es- timated at 20%, and at 70–80% in low trans- mission setting (where prevalence is less than 10%). Evidently, submicroscopic prevalence has increased in areas where control has been successful in reducing transmission (Lindblade et al. 2013). In low transmission setting, the share of asymptomatic cases in transmitting malaria is significant. In regions of seasonal transmission, they can be a source of infec- tion for the new generations of mosquitoes following rain (Sturrock et al. 2013). Active case detection is the intervention recommended for low malaria transmission settings, although there is very little evidence on its efficiency. The most important factors affecting the efficiency of this method are its low sensitivity, inability to detect very low- density infection, continuous importation of parasites, as well as insufficient coverage of the population (Ministry of Health and Med- ical Education 2012) . The most important public health interven- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 172–181 Gh Hassanpour et al.: Asymptomatic Malaria … 179 http://jad.tums.ac.ir Published Online: May 27, 2017 tions aimed at asymptomatic parasitemia are ‘drug mediated strategies’ aimed at asympto- matic parasite reservoirs: 1- mass drug ad- ministration (MDA) in a geographic region - irrespective of infection and symptoms, 2- mass screening and treatment (MSAT) or ag- gressive active case detection, and 3- inter- mittent preventive therapy -repeated treat- ment of high-risk groups. These approaches can be adopted at smaller (modified) scales such as hotspots too. Mathematical models have shown a 15–20% reduction in transmis- sion through the achievement of these goals (Sturrock et al. 2013). To eliminate malaria, in addition to under- scoring the significance of the highest public authorities’ commitment to the program, the World Health Organization has made the fol- lowing recommendations: Programs for detecting and treating patients, disrupting malaria transmission, identification of local foci, establishment of an active sur- veillance system, preventing re-establishment of transmission, and regional cooperation - particularly with neighboring countries to pre- vent the entry of infection into the country. No doubt, the chances of eliminating ma- laria reduce if there are efficient mosquito vectors, special demographic conditions such as suburbanization, low hygiene levels, un- sustainable borders and illegal migrations (Bousema et al. 2012). As a country that has reduced approximately 90% of its cases, Iran is faced with the chal- lenge of disease elimination due to multiple reasons. An important challenge is the issue of cross border importation from Afghanistan and Pakistan. 1.6% (7/446) asymptomatic Afghani immigrants are asymptomatic-infected who can be the potential source of parasites trans- mission (Nateghipour et al. 2011). Based on statistics, over 20% of the treated cases in Iran are patients from Pakistan, a country where there are 4.5 million reported cases of malar- ia, and from which the entries and exits into and from the Eastern borders are not meticu- lously controlled. Moreover, in Iran, different vectors behave differently, which is another challenge of the elimination phase (Hanafi-Bojd et al. 2011). The resistance of the most important vector ie Anopheles stephensi to some insecticides, especially pyrethroids, (Vatandoost and Hanafi- Bojd 2012), as well as the drug resistance of P. falciparum to Chloroquine and some other drugs strongly necessitates an accurate moni- toring to enable the achievement of the elimi- nation program’s goals (Tietje et al. 2014). Conclusions Detection of high-risk foci either it geo- graphically or demographically, is an im- portant strategy for reducing local parasite reservoirs and disrupting the transmission chain. Active and passive methods of case detection must be used to achieve the goal of malaria elimination. In this approach, both groups of clinically symptomatic and asymp- tomatic individuals need to be correctly di- agnosed and treated, at the same time that vector control is under way. Imported malar- ia cases are a potential source of transmis- sion. Thus, targeting symptomatic and asymp- tomatic infections particularly in regions of seasonal and low transmission, keeping in mind that even a small percentage of infected per- sons are sufficient to re-start the malaria trans- mission, demands the accurate monitoring of asymptomatic and submicroscopic cases through highly valid molecular and/or sero- logical methods. Iran is a country with a low burden and limited transmission of malaria, wherein the elimination of local malaria transmission from P. falciparum and the complete elimination of malaria have been targeted for 2015 and 2025, respectively. Most cases of disease how- ever, result from P. vivax, which has special biological characteristics such as, the latent liver stage hypnozoites and the faster im- munity created against it -compared to P. fal- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 172–181 Gh Hassanpour et al.: Asymptomatic Malaria … 180 http://jad.tums.ac.ir Published Online: May 27, 2017 ciparum, thus necessitating the need to take into account other considerations as well (Lindblade et al. 2013). Conducting precise longitudinal follow-up studies on asympto- matic individuals, to examine the possibility of their being gametocytemic, and, to study the period of infection in these individuals in low transmission settings can yield more ac- curate data for correct planning by health policymakers. 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