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Investigation of LAMP Technique… Hadi M. et al 743 ORIGINAL ARTICLE Investigation of LAMP Technique in Diagnosis Type of Plasmodium Species in Anopheles Mosquitoes :A Fast and Practical Technique to Detect Malaria Pathogens in the Field Hadi Mirahmadi1,2, Nadia Kazemipour3, Anis Yazdiani3, Ahmad Mehravaran1,2, Hamid Reza Basseri4, Leili Mohammadi1, Ebrahim Alijani5* ABSTRACT OPEN ACCESS Citation: Hadi Mirahmadi, Nadia BACKGROUND፡ Malaria is one of the main parasitic diseases Kazemipour, Anis Yazdiani, Ahmad and a major health issue in some countries. This study aims to Mehravaran, Hamid Reza Basseri, Leili Mohammadi, Ebrahim Alijani. determine the rate and type of infections of Anopheles mosquitoes Investigation of LAMP Technique in with malaria parasites using the molecular LAMP method in the Diagnosis Type of Plasmodium Species in Anopheles Mosquitoes :A Fast and Southeastern Iran. Practical Technique to Detect Malaria METHODS: In this study, 400 Anopheles mosquitoes were Pathogens in the Field. Ethiop J Health collected by the Zahedan Medical Insecticide Center in Nikshahr Sci. 2021;31 (4):743. doi:http://dx.doi.org/10.4314/ejhs.v31i4.8 City, a high-risk area of malaria transmission in Sistan- Received: September 14, 2020 Baluchestan Province. The mosquitoes were caught manually (by Accepted: December 28, 2020 hand) in domestic (humans and animals), natural, and artificial Published: July 1, 2021 Copyright: © 2021 Hadi M., etal This outdoor places (Shelter pits). After DNA extraction, the LAMP is an open access article distributed method was used, which was compared with Multiplex Nested- under the terms of the Creative Commons Attribution License, which PCR as a standard method. permits unrestricted use, distribution, RESULTS: Out of 400 samples collected from Nikshahr City, 6 and reproduction in any medium, samples (1.5%) were infected with Plasmodium vivax. No provided the original author and source are credited. Plasmodium falciparum or a mix (Plasmodium vivax and Funding: Islamic Azad University, Plasmodium falciparum) was detected in this study. Kerman, Kerman, Iran CONCLUSIONS: The results of this study indicate that in places Competing Interests: The authors declare that this manuscript was with transmission of both species, i.e. Plasmodium vivax and approved by all authors in its form and Plasmodium falciparum, detection of malaria parasites by the that no competing interest exists. Affiliation and Correspondence: LAMP method could be very useful in spotting infections in the 1Infectious Disease and Tropical field. Thus, molecular epidemiological studies could be conducted Medicine Research Center, Resistance annually to monitor malaria in endemic regions. The results of Tuberculosis Institute, Zahedan University of Medical Sciences, this research show that contamination with mosquito malaria Zahedan, Iran. vectors is increasing in Nikshahr City, and it seems that more 2Department of Parasitology and Mycology, Faculty of Medicine, studies will be required to eliminate malaria until 2026. Zahedan University of Medical KEYWORDS: Malaria, Nested-PCR, LAMP, Anopheles Sciences, Zahedan, Iran Mosquitoes 3 Department of Microbiology, Faculty of Basic Sciences, Islamic Azad University, Kerman, Kerman, Iran. INTRODUCTION 4Department of Medical Entomology, School of Public Health, Tehran Malaria is a critical public health issue in tropical and subtropical University of Medical Sciences, regions. In humans, four protozoan parasites, including Tehran, Iran 5Clinical Immunology Research Plasmodium vivax, P. falciparum, P. ovale, and P. malariae lead to Center, Zahedan University of the spread of this mosquito-borne infectious disease. The first Medical Sciences, Zahedan, Iran treatment line for this disease is diverse and depends on *Email: [email protected] Plasmodium species. In other words, proper selection of 744 Ethiop J Health Sci. Vol. 31, No. 4 July 2021 antimalarial drugs is based on thorough species, PCR methods could detect multiple knowledge of pathogens (1-3). Therefore, proper infections. identification of pathogens is important before Independent PCR detection of all pathogens standard chemotherapy. According to a report with corresponding single primers needs more released by the World Health Organization in time, effort, and costs in the case of a mixed 2016, despite various malaria control and infection. Multiplex PCR-based approaches are antimalarial programs, 216 million cases of more effective, in which more than one pair of malaria were reported from 91 countries, which primers are employed in a single response, exceeded the number in 2016 by about 5 million thereby resulting in different sizes of DNA cases. The annual mortality rate of malaria is fragments being amplified simultaneously. In around 445,000 individuals worldwide. Malaria addition, they cut costs, save time, and are deaths declined from 2015 to 2016 in effective in detecting mixed infections. Thus, a Southeastern Asia but increased in Africa, multiplex nested-PCR method has been Eastern Mediterranean, and the Pacific region employed in the present study for simultaneous (4, 5) detection of P. vivax and P. falciparum(12). Iran is one of the known malaria endemic High sensitivity and specificity of this technique places in the Eastern Mediterranean region, with indicate its effectiveness in diagnosing its endemic malaria areas being the provinces of asymptomatic malaria cases of a low parasitic Sistan-Baluchestan and Hormozgan, as well as burden. Besides, it is a perfect method for Jiroft and Kahnuj Cities in Kerman Province (6) detecting malaria in low-transmission areas, In Iran, the main goal of the malaria elimination which could be used in monitoring drug program is to stop local transmission of the resistance as well. Furthermore, many samples disease and to become a malaria-free region in could be tested simultaneously (12). 2026(7). One of the essential strategies in the Loop-mediated isothermal amplification Global Malaria Control Program is the control of (LAMP) has made it possible to use a specific, Anopheles mosquitoes, which is considered one rapid, sensitive, and simple technique for of the most effective ways to eradicate and diagnosing a variety of diseases, including reduce malaria transmission around the world. vector-borne ones (15). Bst DNA polymerase Besides, awareness of the diversity of Anopheles and a set of four specially designed primers, species and infection with sporozoites in their which identify six separate regions of the target salivary glands is one of the most decisive DNA, are employed by LAMP. The target gene epidemiological steps in planning malaria could be amplified and detected in an isothermal control and prevention methods appropriate for phase (15-17).Upon accumulation of about 10 9 local conditions (8)Various biochemical, copies of target DNA in less than one hour, molecular, and immunological methods(9-11), auto-cycling strand displacement DNA synthesis such as isoenzyme electrophoresis, polymerase is performed. A series of stem-loop DNA chain reaction (PCR), and monoclonal structures of different lengths are included in antibodies have been used to detect Plasmodium amplified products (18,19). Simple detection in mosquitoes(12, 13). One of the most common could be performed by visually inspecting detection methods for malaria parasites in turbidity of magnesium pyrophosphate, i.e. a Anopheles mosquitoes is dissection of salivary byproduct of DNA synthesis, produced in glands in female Anopheles, captured by proportion to the amount of amplified DNA (20, stereomicroscopy and light microscopy. 21). Using a loopamp real-time turbid meter, However, this method is very cumbersome, real-time detection could be performed as well time-consuming, and lowly sensitive (12,14). (22). Due to the relatively favorable climate and PCR methods are highly sensitive and geographical conditions in the southern and specific, capable of detecting 1–10 parasites per southeastern regions of Iran as well as migration microliter of blood, being five times more to and from Afghanistan and Pakistan, malaria is effective than microscopic detection by Giemsa- a major public health issue in Iran .Sistan- stained spreads. In addition to detecting parasite Baluchestan Province has a long border with Pakistan, across which the largest number of Investigation of LAMP Technique… Hadi M etal 745 malaria cases is observed (23). This study aims fed Anopheles mosquitoes were to examine the rate and type of Anopheles captured (Figure 1). The Anopheles mosquitoes mosquito infections with sporozoites by were caught manually (by hand) in domestic Multiplex Nested-PCR and LAMP, which is (humans and animals), natural, and artificial performed in Nikshahr City of Sistan- outdoor sites (Shelter pits), with suction tubes or Baluchestan Province. aspirators utilized. All aspirating specimens were killed by a small chloroform-impregnated METHODS cotton pad placed in plastic cups. Just after the collection task, the samples were placed in 70% Sample preparation: This descriptive cross- alcohol-containing tubes and transferred to the sectional study was conducted in the time period relevant health center. Using a high from March 2018 to July 2019 in Nikshahr City, magnification entomology loop, the blood-fed a high-risk area of malaria transmission in Anopheles mosquitoes were separated and Sistan-Baluchestan province. Populations transferred into 1.5 ml tubes. Next, each sample surveyed in the city included Bennett, Fanuj, was assigned
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