Abstract
Background:
Malaria elimination program has been launched in Iran with the technical support of the World Health Organization since 2009. To achieve the goal of malaria eradication, not only all positive cases should be diagnosed and treated promptly but also treated patients might be considered asymptomatic reservoirs in the establishment of the malaria transmission cycle.Objectives:
The present study aimed to follow up and monitor malaria-treated cases using sensitive molecular tools, as well as microscopic and rapid diagnostic test (RDT) techniques, in Bashagard district, Hormozgan province, Iran.Methods:
This cross-sectional survey was conducted in Bashagard district of Hormozgan province for 12 months from 2015 to 2016. A total number of 208 malaria cases treated according to the national malaria treatment guideline (I.R. Iran, 3rd edition) were randomly selected from databases to be evaluated for Plasmodium infection using microscopic, RDT, and nested-PCR (using 18ssrRNA) techniques.Results:
Of the total number of 208 participants in the study, 39.9% were male and 61.1% were male. None of the participants had symptoms of malaria before sampling. Based on the results of microscopic methods, RDT, and molecular analysis for the detection of malaria parasites, no positive malaria treated cases were found.Conclusions:
The results of the study showed that a robust malaria surveillance system including screening, diagnosis, timely treatment, and follow-up of treated cases plays an important role in the malaria elimination program to be implemented successfully.Keywords
Malaria Elimination Program Treated Cases Plasmodium Nested-PCR Iran
1. Background
Malaria is the most important vector-borne infectious and parasitic disease, which is transmitted through mosquito bites. Malaria transmission occurs in many parts of the world, threatening more than 40% of the world’s population. Indeed, about 97 countries in the world are considered as malaria-endemic areas. According to the World Health Organization (WHO), about 216 million malaria cases occurred worldwide in 2016, causing 445,000 deaths (1). Iran is considered one of the malaria-endemic countries of the Middle East region. Malaria is one of the most important health problems in the southern and southeastern parts of the country, including Sistan and Baluchistan, Hormozgan, and Kerman provinces. The dominant species of malaria in Iran is Plasmodium vivax, while P. falciparum has been reported in 10 - 15% of the cases (2, 3). Anopheles stephensi and An. culicifacies are the main vectors of malaria in Iran. Furthermore, An. superpictus, An. sacharovi, An. dthali, An. pulcherrimus, and An. fluviatilis species complex is responsible for malaria transmission in Iran (4, 5).
The impact of malaria on public health and health promotion elements, as well as its social and economic effects on communities’ development, has caused international organizations to focus on this disease, ultimately codifying a comprehensive plan for control, elimination, and eradication of malaria (6, 7). In recent years, the number of malaria cases has decreased significantly in Iran (11,460 to 705 cases during 2008 to 2016) (1). Because of the successful implementation of malaria prevention and control programs in Iran and the subsequent reduction of malaria cases in recent years, the malaria elimination program has been launched in the country with technical support from the WHO since 2009 (2, 7, 8). The main goal of the malaria elimination plan is to stop malaria transmission locally. To achieve the goal of malaria elimination, all positive cases should be promptly diagnosed and treated, especially asymptomatic and low parasitic ones that cannot be detected by routine procedures (9).
Following the development of novel and sensitive methods for malaria detection, especially molecular techniques, asymptomatic malaria cases are now detectable (10). Nonetheless, treated patients might act as an asymptomatic reservoir in the establishment of the malaria transmission cycle (11). Many studies have shown that asymptomatic carriers, as unknown reservoirs, play important roles in establishing the malaria transmission cycle in different parts of the world (12). Because of the importance of this issue in the malaria elimination program, it attracted the attention of several researchers and led to many reports about the presence of asymptomatic cases in Africa, South America, and Asia (12), including Nigeria (13), Zambia (14), Gabon (15), Senegal (16), Brazil (17), Colombia (18), Yemen (19), Thailand (20), Cambodia (21), Solomon Islands (10), and Minab district of Iran (2). On the contrary, some studies have shown no cases of asymptomatic malaria cases in Sri Lanka and Iran (3, 22-24).
2. Objectives
The present study aimed to follow up and monitor malaria treated cases using sensitive molecular tools (nested-polymerase chain reaction (PCR)), as well as microscopic and rapid diagnostic test (RDT) techniques in Bashagard district, Hormozgan province, Iran. The results of this study would facilitate the implementation of malaria elimination programs in Hormozgan province and the entire country.
3. Methods
3.1. Study Area
Hormozgan province is located in the southern part of Iran and is considered an endemic area for malaria, accounting for 11.5% (81 of 705 cases) of all Iranian malaria cases in 2016 (Figure 1). This cross-sectional survey was conducted in Bashagard district of Hormozgan province [(26 ° 21’58 ”N 57 ° 35’48 ”E); area = 9,200 km2] during 12 months from 2015 to 2016. According to the 2017 census, the population of Bashagard district was 35931 in 8615 families. Its average annual relative humidity is 46.2% and the average annual precipitation is about 150 mm. In addition, the daily temperature varies from 7.7°C to 44.2°C in this district (www.weather.ir). These features have created a good condition for malaria transmission in this region throughout the year. Currently, the malaria elimination program is ongoing in Bashagard district (API < 1/1000) (23).
Iran’s map indicating the study area; Hormozgan province, Bashagard district
3.2. Sampling Methods
Five hundred people were randomly selected from the entire population of Bashagard district. After the initial review, 208 out of 500 were found as the treated cases of malaria. All 208 cases (125 women and 83 men) were included in the study. The cases had been treated according to the National Malaria Treatment Guideline (I.R. Iran, 3rd edition). The selected cases were evaluated for Plasmodium infection rate by using microscopic, RDT, and nested-PCR (using 18ssrRNA) techniques. The inclusion criteria of the study were having a history of malaria treatment and not having the symptoms and clinical signs of malaria. Individuals with a history of travel to other malaria-endemic areas in the past year were excluded from the study. Before starting the project, all participants signed consent forms and agreed to complete the trial. After the interviews and registration of the profiles, 3 mL of blood samples were taken from each participant for the diagnosis of the malaria parasite.
3.3. Microscopy
The microscopic method is the gold standard for the detection of malaria (25-28). Briefly, after scrubbing the finger with 70% ethanol, a small scratch was created by a lancet. Then, thick and thin blood smears were prepared from each person according to the standard method. All the slides were stained with Giemsa method. After drying, the thin smear was fixed with methanol; the slides were stained with Giemsa 10% for 20 minutes and examined at 1000x immersion oil for the diagnosis of malaria parasite by expert microscopists (29). The RDT was done in the field. After transferring samples to the laboratory, nested PCR and microscopic test were done by an experienced person blind to the results of the RDT. In order to conduct the follow-up, 30 and 60 days after the initial sampling, new blood smears were taken and examined for Plasmodium using the microscopic test.
3.4. Rapid Diagnostic Test
The rapid diagnostic test is an immuno-chromatographic experiment, which recognizes the presence of a specific malaria antigen in the blood sample. The kit has a two-line strip previously coated with two monoclonal antibodies, one against the pan-specific lactate dehydrogenase (pLDH) of all Plasmodium species and the other against P. falciparum histidine-rich protein 2 (HRP2). In this study, 5 µL of each patient’s blood sample was added to the sample well in the test card. Then, 60 µL of the buffer solution was added to the well. After about 15 minutes, the formation of specific bands showed if the patient was infected with human malaria species (29). In so doing, the Combo test kit was used (Premier Medical Corporation Ltd., Mumbai, India) and the test was performed according to the manufacturer’s instructions.
3.5. Nested-PCR
In this study, the nested-PCR method was used to diagnose Plasmodium species. This method benefits from high sensitivity and accuracy in detecting asymptomatic cases (30). In this study, the Promega kit was used to extract DNA from blood samples (Promega, Madison, WI, USA). Additionally, the molecular diagnostic method was carried out based on research by Snounou et al. (31). Positive and negative controls were tested in each series of reactions. Accordingly, a sample was considered positive for P. vivax and P. falciparum if a 120- and 205-base-pair fragment was identified, respectively (2, 25).
4. Results
To investigate the presence of asymptomatic malaria reservoirs in Bashagard district, 208 malaria treated cases with no specific malaria symptoms were included in this study. The demographic characteristics of the participants are shown in Table 1. Of the total number of 208 participants in the study, 39.9% were male and 61.1% were male. None of these participants had symptoms of malaria before sampling. Then, they were assessed using microscopic, RDT, and nested-PCR methods, the results of which are given as follows. All of them had a history of P. vivax and their treatments were based on the national malaria treatment protocol. The results of all microscopic slides were negative. In order to increase the accuracy of the results, all microscopic slides were re-examined by an expert microscopist. The RDT was used as a complementary method for all 208 samples. The results of all samples were negative using the RDT kits. Despite the use of a sensitive molecular method for all specimens, no positive cases were found in the nested-PCR method (Figure 2).
Distribution of the Sample Based on Age Groups, Sex, and History of Malaria Infection
| Parameter | No. (%) |
|---|---|
| Sex | |
| Female | 125 (61.1) |
| Male | 83 (39.9) |
| Total | 208 (100) |
| Age group | |
| 5 - 20 | 83 (39.9) |
| 21 - 35 | 46 (22.12) |
| 36 - 50 | 57 (27.4) |
| 51 - 65 | 22 (10.58) |
| Total | 208 (100) |
| Malaria history | |
| Yes | 208 (100) |
| No | 0 |
| Total | 208 (100) |
The results of PCR assay on human samples stained with ethidium bromide on 1.2% agarose gel; samples (lanes 1, 2, and 8), P. vivax reference control (lane 4), negative control (lanes 3 and 7), P. falciparum reference control (lane 6), and molecular marker (100 bp Cinnagen, Iran) (lane 5).
5. Discussion
Malaria elimination is the common goal of WHO and Iran’s health system. This study was conducted in accordance with the malaria elimination program in Iran. Despite the use of a sensitive molecular technique, microscopic methods, and RDT, there were no cases of asymptomatic malaria among treated cases in Bashagard district. In recent years, local malaria transmission has decreased in Iran and according to the WHO classification, Iran is a candidate for the malaria elimination phase (API < 1/1000) (7). In the elimination phase, all effective factors in creating a malaria transmission cycle in the region should be considered. Besides, all positive cases must be diagnosed and timely treated, especially low parasite and asymptomatic cases that are not detectable by routine methods. Moreover, malaria treated cases can act as asymptomatic reservoirs in malaria-endemic areas and consequently, they should be taken into consideration (11). In the malaria elimination program, in addition to microscopic and RDT methods, more robust and sensitive diagnostic techniques, including tools to enable the detection of parasite carriers (low parasitism, asymptomatic infections) are required. In this study, microscopic, serology, and molecular methods were used simultaneously to increase sensitivity in the diagnosis of Plasmodium species.
The findings showed that because of the great surveillance system for case finding, diagnosis, and treatment of malaria cases, there were no positive malaria treated cases in Bashagard district. It can be concluded that the timely and appropriate treatment of malaria cases, as well as monitoring of the treatment process, is one of the key strategies for the control and elimination of malaria. These results supported those of the previous investigations in Bashagard district, reporting no cases of asymptomatic malaria (3, 23, 24). The results were also in agreement with those of a study performed by Amirshekari et al. in Kerman province, which revealed no asymptomatic malaria cases among indigenous people (26). In contrast, the study by Shahbazi et al. showed two asymptomatic carriers of malaria in treated cases in Bashagard and Minab districts (11). Another survey in Minab district also indicated some cases of asymptomatic malaria. The difference between the study results might be attributed to differences in the monitoring of health systems, as well as to specific climatic and environmental conditions (2).
Several reports have demonstrated the high prevalence of asymptomatic malaria in the world, especially in Central and South America, Africa, and East Asia, which is not aligned with the present study results. This contradiction might have resulted from genetic diversity in humans, parasites, and carriers of malaria, different weather conditions, demographic characteristics, living conditions, population movements, and differences in surveillance systems (27, 28). The strengths of this study included its high sensitivity and accuracy of diagnosis resulted from the proper sample size and using a sensitive molecular method along with microscopic and RDT methods. However, the study limitations were related to field sampling and following up of cases due to the displacement of the study population.
5.1. Conclusions
It can be claimed that the robust malaria surveillance system (detection, diagnosis, and treatment of positive cases of malaria and monitoring of the treatment process) has caused the malaria elimination program to be successfully implemented in Bashagard district. According to the malaria elimination program, a series of research is necessary for the study region to monitor and evaluate asymptomatic malaria in high-risk areas.
Acknowledgements
References
-
1.
World Health Organization. World Health Organization (WHO) malaria report 2017. World Health Organization Press; 2017.
-
2.
Turki H, Raeisi A, Malekzadeh K, Ghanbarnejad A, Zoghi S, Yeryan M, et al. Efficiency of nested-PCR in detecting asymptomatic cases toward malaria elimination program in an endemic area of Iran. Iran J Parasitol. 2015;10(1):39-45. [PubMed ID: 25904944]. [PubMed Central ID: PMC4403538].
-
3.
Turki H, Rashid G, Shekari M, Raeisi A, Sharifi-Sarasiabi K. Malaria elimination program: Absence of asymptomatic malaria and low parasitic in endemic area of Rudan district, Hormozgan province, Iran. Hormozgan Med J. 2017;21(4):225-31. https://doi.org/10.29252/hmj.21.4.225.
-
4.
Nateghpour M, Turki H, Keshavarz H, Edrissian GH, Mohebali M, Rahimi Foroushani A. A parasitological and serological study in malaria suspected patients in Hormozgan province, southeastern Iran. Iran Red Crescent Med J. 2010;12(3):242-6.
-
5.
Hanafi-Bojd AA, Vatandoost H, Oshaghi MA, Charrahy Z, Haghdoost AA, Zamani G, et al. Spatial analysis and mapping of malaria risk in an endemic area, south of Iran: A GIS based decision making for planning of control. Acta Trop. 2012;122(1):132-7. [PubMed ID: 22245147]. https://doi.org/10.1016/j.actatropica.2012.01.003.
-
6.
Global Malaria Programme. World malaria report 2014. Geneva: World Health Organization; 2014.
-
7.
Raeisi A, Gouya MM, Nadim A, Ranjbar M, Hasanzehi A, Fallahnezhad M, et al. Determination of malaria epidemiological status in Iran's malarious areas as baseline information for implementation of malaria elimination program in Iran. Iran J Public Health. 2013;42(3):326-33. [PubMed ID: 23641411]. [PubMed Central ID: PMC3633804].
-
8.
Elimina M. Malaria elimination, guide for participants. World Health Organization; 2016.
-
9.
Sturrock HJ, Hsiang MS, Cohen JM, Smith DL, Greenhouse B, Bousema T, et al. Targeting asymptomatic malaria infections: Active surveillance in control and elimination. PLoS Med. 2013;10(6). e1001467. [PubMed ID: 23853551]. [PubMed Central ID: PMC3708701]. https://doi.org/10.1371/journal.pmed.1001467.
-
10.
Harris I, Sharrock WW, Bain LM, Gray KA, Bobogare A, Boaz L, et al. A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: Challenges for malaria diagnostics in an elimination setting. Malar J. 2010;9:254. [PubMed ID: 20822506]. [PubMed Central ID: PMC2944341]. https://doi.org/10.1186/1475-2875-9-254.
-
11.
Shahbazi A, Farhadi P, Yerian M, Bazmani A, Nakhjiri SK, Rasouli A, et al. Detection of asymptomatic carriers of plasmodium vivax among treated patients by nested PCR method in Minab, Rudan and Bashagard, Iran. Iran J Parasitol. 2013;8(4):586-92. [PubMed ID: 25516740]. [PubMed Central ID: PMC4266123].
-
12.
Lindblade KA, Steinhardt L, Samuels A, Kachur SP, Slutsker L. The silent threat: Asymptomatic parasitemia and malaria transmission. Expert Rev Anti Infect Ther. 2013;11(6):623-39. [PubMed ID: 23750733]. https://doi.org/10.1586/eri.13.45.
-
13.
Eke R, Chigbu L, Nwachukwu W. High prevalence of asymptomatic Plasmodium infection in a suburb of Aba Town, Nigeria. Ann Afr Med. 2006;5(1):42-5.
-
14.
Stresman GH, Kamanga A, Moono P, Hamapumbu H, Mharakurwa S, Kobayashi T, et al. A method of active case detection to target reservoirs of asymptomatic malaria and gametocyte carriers in a rural area in Southern Province, Zambia. Malar J. 2010;9:265. [PubMed ID: 20920328]. [PubMed Central ID: PMC2959066]. https://doi.org/10.1186/1475-2875-9-265.
-
15.
Nkoghe D, Akue JP, Gonzalez JP, Leroy EM. Prevalence of Plasmodium falciparum infection in asymptomatic rural Gabonese populations. Malar J. 2011;10:33. [PubMed ID: 21306636]. [PubMed Central ID: PMC3041723]. https://doi.org/10.1186/1475-2875-10-33.
-
16.
Vafa M, Troye-Blomberg M, Anchang J, Garcia A, Migot-Nabias F. Multiplicity of Plasmodium falciparum infection in asymptomatic children in Senegal: Relation to transmission, age and erythrocyte variants. Malar J. 2008;7:17. [PubMed ID: 18215251]. [PubMed Central ID: PMC2267475]. https://doi.org/10.1186/1475-2875-7-17.
-
17.
Alves FP, Durlacher RR, Menezes MJ, Krieger H, Silva LH, Camargo EP. High prevalence of asymptomatic Plasmodium vivax and Plasmodium falciparum infections in native Amazonian populations. Am J Trop Med Hyg. 2002;66(6):641-8. [PubMed ID: 12224567]. https://doi.org/10.4269/ajtmh.2002.66.641.
-
18.
Cucunuba ZM, Guerra AP, Rahirant SJ, Rivera JA, Cortes LJ, Nicholls RS. Asymptomatic Plasmodium spp. infection in Tierralta, Colombia. Mem Inst Oswaldo Cruz. 2008;103(7):668-73. [PubMed ID: 19057816]. https://doi.org/10.1590/S0074-02762008000700007.
-
19.
Bin Mohanna MA, Bin Ghouth AS, Rajaa YA. Malaria signs and infection rate among asymptomatic schoolchildren in Hajr Valley, Yemen. East Mediterr Health J. 2007;13(1):35-40. [PubMed ID: 17546903].
-
20.
Congpuong K, Saejeng A, Sug-Aram R, Aruncharus S, Darakapong A, Meshnick SR, et al. Mass blood survey for malaria: Pooling and real-time PCR combined with expert microscopy in north-west Thailand. Malar J. 2012;11:288. [PubMed ID: 22909399]. [PubMed Central ID: PMC3478204]. https://doi.org/10.1186/1475-2875-11-288.
-
21.
Hoyer S, Nguon S, Kim S, Habib N, Khim N, Sum S, et al. Focused screening and treatment (FSAT): A PCR-based strategy to detect malaria parasite carriers and contain drug resistant P. falciparum, Pailin, Cambodia. PLoS One. 2012;7(10). e45797. [PubMed ID: 23049687]. [PubMed Central ID: PMC3462177]. https://doi.org/10.1371/journal.pone.0045797.
-
22.
Fernando SD, Abeyasinghe RR, Galappaththy GN, Rajapaksa LC. Absence of asymptomatic malaria infections in previously high endemic areas of Sri Lanka. Am J Trop Med Hyg. 2009;81(5):763-7. [PubMed ID: 19861607]. https://doi.org/10.4269/ajtmh.2009.09-0042.
-
23.
Turki H, Zoghi S, Mehrizi AA, Zakeri S, Raeisi A, Khazan H, et al. Absence of asymptomatic malaria infection in endemic area of bashagard district, Hormozgan province, Iran. Iran J Parasitol. 2012;7(1):36-44. [PubMed ID: 23133470]. [PubMed Central ID: PMC3488819].
-
24.
Zoghi S, Mehrizi AA, Raeisi A, Haghdoost AA, Turki H, Safari R, et al. Survey for asymptomatic malaria cases in low transmission settings of Iran under elimination programme. Malar J. 2012;11:126. [PubMed ID: 22533733]. [PubMed Central ID: PMC3464154]. https://doi.org/10.1186/1475-2875-11-126.
-
25.
Motevalli Haghi A, Delavari M, Nateghpour M, Shekari M, Turki H, Raeisi A, et al. Asymptomatic malaria control program in high-risk Jask district, Hormozgan province, Iran. Sci J Sch Pub Health Inst Pub Health Res. 2015;13(2):95-103.
-
26.
Amirshekari MB, Nateghpour M, Raeisi A, Motevalli Haghi A, Farivar L, Edrissian G. Determination of asymptomatic malaria among Afghani and Pakistani immigrants and native population in south of Kerman province, Iran. Iran J Parasitol. 2016;11(2):247-52. [PubMed ID: 28096860]. [PubMed Central ID: PMC5236103].
-
27.
Tanner M, Greenwood B, Whitty CJ, Ansah EK, Price RN, Dondorp AM, et al. Malaria eradication and elimination: Views on how to translate a vision into reality. BMC Med. 2015;13:167. [PubMed ID: 26208740]. [PubMed Central ID: PMC4514994]. https://doi.org/10.1186/s12916-015-0384-6.
-
28.
Mirahmadi H, Spotin A, Fallahi S, Taghipour N, Turki H, Seyyed Tabaei SJ. Cloning and sequence analysis of recombinant plasmodium vivax merozoite surface protein 1 (PvMSP-142 kDa) In pTZ57R/T vector. Iran J Parasitol. 2015;10(2):197-205. [PubMed ID: 26246817]. [PubMed Central ID: PMC4522295].
-
29.
Wongsrichanalai C, Barcus MJ, Muth S, Sutamihardja A, Wernsdorfer WH. A review of malaria diagnostic tools: Microscopy and rapid diagnostic test (RDT). Am J Trop Med Hyg. 2007;77(6 Suppl):119-27. [PubMed ID: 18165483]. https://doi.org/10.4269/ajtmh.2007.77.119.
-
30.
Ebrahimzadeh A, Nouri Dalir S, Mirahmadi H, Mehravaran A, Salimi Khorashad A, Turki H. The incidence of current infection with different human malaria species by polymerase chain reaction for diagnosis of suspicious malaria patients on elimination region Sistan and Baluchistan province, southeast of Iran. Jundishapur J Microbiol. 2017;10(10). https://doi.org/10.5812/jjm.58254.
-
31.
Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol. 1993;58(2):283-92. [PubMed ID: 8479452]. https://doi.org/10.1016/0166-6851(93)90050-8.