Investigation of LAMP Technique in Diagnosis Type of Plasmodium

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

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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 a three-letter code and then stored Lashar, Ahuran, and Markazi sampled from in the tubes at -20 °C until DNA extraction. selected villages every 15 days, in which blood-

Figure 1: Geographical locations where this study was carried out

Multiplex Nested-PCR Method: DNA The amplified products were electrophoresed on extraction was performed by the column method 2% agarose gels in Tris–borate-EDTA for P. through employing a DNA extraction kit vivax and P. falciparum and then by the (Produced by the DynaBio Blood/Tissue ultraviolet Trans illuminator stained with a red Genomic DNA Extraction Kit). Multiplex gel for visual detection. Nested-PCR was performed to determine species The PCR steps included denaturation of the of Plasmodium via employing designed primers DNA molecule at 95 °C for 5 min and at 94 °C (Table 1). A pair of oligonucleotide primers for 30 s, with 25 cycles for the first step and 30 (rPLU6 and rPLU5) was employed for the PCR- cycles for the Nested-PCR, annealing at 58 °C 1 reaction in this study. In this reaction, primers, for 1 min, extension at 72 °C for 60 s for the including rFAL.1, rFAL.2, rVIV.1, and rVIV.2 first step and 30 seconds for the Nested-PCR, were used to simultaneously determine final extension at 72 °C for 10 min for the first Plasmodium species (13, 24). The first PCR step and 5 min for the Nested-PCR, and finally, product was diluted at a 1:100 ratio with completion of the reaction and a reduction in the distilled water as a template in the second PCR. temperature to 20 °C (22,25,26).

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Table 1: Sequences of the primers used in the study for representation of the Plasmodium ssrRNA genes and multiplex/nested PCR protocol

LAMP reaction: Table 2 provides information provided as a master mix. Next, 24 µl of the about the LAMP primer for amplifying gene mixture was poured separately into the new coding for 18S rRNA specific Plasmodium microtubes, and 1 µl of each sample’s DNA was genus (27, 28). The LAMP reaction mixture (25 added next to each microtube. The reaction µL) included 1 µl of template DNA (100 ng/µL), mixture was incubated in a water bath for 60 40 pmol of each of the FIP (Forward Internal) min at 60 °C and inactivated for 2 min at 80 °C. and BIP (Backward Internal) primers, 20 pmol SYBR Green I (Invitrogen Carlsbad, CA, USA) of primer LB (Loop Backward), 5 pmol of each was added to the reaction microtubes, and the of the F3 (Forward 3) and B3 (Backward 3) resulting amplicons were spotted by visual primers, 8 U Bst2 DNA polymerase (New observation. By triggering the reactions for 30, England Biolabs, Ipswich, MA, USA), 2 × 45, 60, and 75 min at 60–64 °C in duplicate, reaction buffer of 40 mM Tris–HCl (pH 8.8), 20 optimal time and temperature conditions for the mMKCl, 20 mM (NH4)2SO4, 16 mM MgSO4, LAMP assay were defined. Besides, a LAMP 0.2% Tween 20, 0.8 M betaine (Sigma-Aldrich), reaction would be considered positive for as well as 1.4 mM deoxynucleoside Plasmodium spp if naked eyes detected an triphosphates (dNTP) according to Table 3. apparent rise in turbidity as opposed to the The amount of reagents was computed based negative control. Two researchers assessed the on the total number of the samples, which was results blindly.

Investigation of LAMP Technique… Hadi M etal 747

Table 2: Sequences of the LAMP primer used in the study for representation of the 18S rRNA specific Plasmodium genus

Table 3: LAMP reagents for both Plasmodium species

Statistical analysis: Diagnostic performances of consistency of the results among the diagnostic the LAMP method were evaluated using tools, the Kappa value was defined. multiplex/nested-PCR as the standard method. SPSS 16.0 (SPSS Inc., Headquarters USA) was employed to perform the analyses. To establish

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RESULTS gel, Lane1: Positive control of Plasmodium falciparum (205 bp), Lane2: Positive control of Multiplex Nested-PCR: In the present study, Plasmodium viv(120 bp), Lane3: Positive the samples (400 blood-fed Anopheles control of mixed infections, Lane4: Ladder 100 mosquitoes) were collected within the time bp, Lanes 5,6,7: P. vivax period from March 2018 to July 2019 in a high- risk region of malaria transmission in Sistan- LAMP reaction: Upon addition of SYBR Green Baluchestan Province, Nikshahr City. Out of I to the reaction microtubes, the positive LAMP 400 samples, 6 (1.5%) samples were infected reaction was visually detectable; in the LAMP with Plasmodium vivax via Multiplex Nested- technique, the positive reaction turned green, PCR. In this method, no infection with and the negative one remained orange (Figs. 3a Plasmodium falciparum and the mix and 3b). Besides, the positive LAMP reaction (Plasmodium vivax and Plasmodium falciparum) produced a typical ladder-like pattern of was observed (Figure 2). multiple bands of various sizes until well loaded on a 2% agarose gel stained with a red gel (Figure 3c). When the LAMP reaction time and temperature were kept fixed for 90 min at 60 °C for the 18SSU rRNA genomic target, the optimum results were achieved. Similar to the Multiplex Nested-PCR method, 8 (2%) out of 400 samples were infected with Plasmodium vivax by the LAMP technique. Like the previous method, no infection with Plasmodium falciparum or the mix (Plasmodium vivax and Plasmodium falciparum) was observed. Figure 2: Electrophoresis of the ssrRNA gene According to Kappa measure of agreement, both multiplex/nested PCR products on a 2% agarose methods were completely consistent with each other (К=1).

Figure 3: Visualization of LAMP products with SYBR Green I a under natural light, b under UV light and c agarose gel electrophoresis. N no template control, P positive control

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DISCUSSION In a similar study in India, 523Anopheles mosquitoes were studied to detect infection with Malaria is still a leading health issue in the Plasmodium. The results of this study showed southeastern regions of Iran, especially in that Anopheles annularis was the predominant Sistan-Baluchestan Province. In addition, it is species, yet no sporozoites were observed after effective in preventing economic and social salivary gland separation and microscopic growth in these deprived regions (29). In the examination. However, 7 Anopheles annularis present study, two molecular methods, including mosquitoes (3.4%) were infected with Multiplex Nested-PCR and LAMP, were used Plasmodium falciparum by PCR (34). In another for the first time in Iran to detect malaria study in the Western India, Malaria infection in infections in vector mosquitoes. The mentioned Anopheles was investigated by the Nested-PCR two methods were highly sensitive, identified method, according to which, 2.8% of Anopheles the genus and species of the parasites, and could subpictus were found to be contaminated with a detect mix infections. The current study was mix of Plasmodium falciparum and vivax (35). conducted in Nikshahr City, one of the high-risk Many factors influence prevalence of areas of malaria transmission in Sistan- Plasmodium species in different regions of the Baluchistan Province. In this study, a total of world. Climatic and ecological differences could 400 blood-fed female Anopheles mosquitoes be very effective in this regard. Determining the were captured. The results of this study showed composition of Anopheles species and the that the dominant species of Anopheles amount of sporozoites in their salivary glands by mosquitoes in the studied area was A. reliable and sensitive methods is crucial for culicifacies (83.33%). malaria prevention plans (8). Plasmodium vivax is the most prevalent This study shows that the Multiplex malaria species in Iran, which is confirmed by Nested-PCR method could be effectively used contamination of 1.5% of the mosquitoes in for a large number of samples. This procedure Nikshahr City, being in line with findings of does not require to dissect salivary glands, yet it other studies (30). A similar study was can even examine dried and old specimens. conducted in Chabahar and Iranshahr by Therefore, there is no need for equipping Oshaghi et al (2004), in which Plasmodium laboratories and dispatching personnel to vivax was found to be the predominant species malaria endemic regions because samples in the captured blood-fed female mosquitoes collected in these regions could be tested at (31). central laboratories. On the other hand, the Assmar et al (2005) conducted a study in LAMP method employed for the first time in endemic regions of Iran using PCR, and the Iran to detect malaria in mosquitoes is simple, results of which showed that Plasmodium vivax sensitive, and practical, which needs no was the dominant parasite in the studied regions expensive facilities and equipment. Besides, it (32). Kalantari et al (2017) conducted a study in could be used with a thermos of boiling water to Fars Province, in which PCR identified a total of easily detect contaminants in the field without 512 female Anopheles mosquitoes. The results wasting any time. showed that Anopheles dthali (71.38%) was the Afghanistan and Pakistan are recognized as dominant species in the mentioned region, yet the most dangerous countries for malaria no Plasmodium parasite infection was reported transmission. Accordingly, since a large number by mosquito PCR. However, in the present of job seekers in Sistan-Baluchestan Province, study, infections (1.66%) were reported in especially in NikShahr City, are from these two Anopheles mosquitoes. Sistan-Baluchestan countries, Iran remains on the list of malaria- Province is one of the endemic regions of endemic countries (3, 30, 36). In a study in malaria, with the difference between its endemic Konarak County, the reason for focal changes of status and that of Fars province having caused malaria and its increasing incidence in 2008 was the present study to report Plasmodium parasitic found to be presence of aliens (37). In another infections in A. stephensi (33). research, Raisi et al. reported that most non-

750 Ethiop J Health Sci. Vol. 31, No. 4 July 2021 indigenous malaria cases in Iran's Sistan- must be conducted annually to monitor this Baluchestan Province were emanated from disease in endemic regions. According to the bordering cities of Baluchestan State of Pakistan studies mentioned above and the present one, (38). In addition, Poudat et al. investigated malaria vector mosquito infection is increasing factors affecting a malaria reduction in Bandar in Nikshahr City. Given that Sistan-Baluchestan Abbas City and concluded that the natives of Province is in the course of eliminating malaria, that city were always at the risk of malaria failure to control this disease could lead to an epidemics due to the presence of immigrants and epidemic. Accordingly, continuous and movement of aliens (39). Therefore, the substantial control measures are required to be importance of migration as a major source of the implemented in all regions of the country. crisis and a major challenge in controlling Furthermore, all border entry points need to be malaria should be considered. controlled, with illegal immigrants required to Epidemiological investigations show that be prevented from entering the country. malaria in Iran, unlike that in Africa and other countries, is subject to climatic conditions and ACKNOWLEDGMENTS on the verge of eradication (25, 26). This implies The authors are grateful to the Faculty of that this unstable state could contribute to the Medicine, Zahedan University of Medical concern that increased rainfalls in high-risk Sciences, Zahedan, Iran. regions could be associated with the higher prevalence of the disease (40). Climatic REFERENCES conditions, socioeconomic and cultural factors, quantity and quality of malaria control 1. Mehravaran A, Mirahmadi H, Mohamadi L, programs, common borders with Afghanistan Dahmardeh H. Traditional herbal treatment for and Pakistan, and issues related to Afghan and plasmodium falciparum: A systematic review Pakistani immigrants are causative factors in the on traditional plants to treat malaria in Iran. annual incidence of the disease in Sistan- International Journal of Psychosocial Baluchestan Province (36). For the reasons Rehabilitation. 2020;24(5):7469-81. mentioned above and due to the fact that Sistan- 2. Mirahmadi H, Yazdani A, Fallahi S. Baluchestan province is in the course of Determination of the prevalence and type of plasmodium in mosquitoes present in the city eliminating malaria, one of the most effective of Nik Shahr, Southern Sistan and Baluchestan ways to monitor the abovementioned program is Province, IR Iran, by Multiplex Nested-PCR. infection observation in Anopheles mosquitos. Yafteh. 2019;20(4). The results of this study, as against past studies, 3. Organization WH. World malaria report 2015: showed that malaria vector mosquito infection in World Health Organization; 2016. Nikshahr City was increasing. Furthermore, it 4. Mirahmadi H, Safari T, Metanat M, seems that elimination of malaria until 2026 Mehravaran A, Raeghi S. Sequence Analysis requires further research. of Pvama-1 among Plasmodium Vivax The results show that in places with Isolates in Sistan-Baluchistan. Ethiopian transmission of both species, i.e. Plasmodium Journal of Health Sciences. 2020;30(4). vivax and Plasmodium falciparum, multiplex 5. Hadi Mirahmadi, AK, AK, NS, SMT, nested-PCR could be very effective in Mohamad Kazem Momeni, et al. diagnosing malaria parasites for detection of Retrospective Epidemiological Study of mixed cases of the disease in both humans and Malaria from 1999 to 2016 in Khash, Iran: A vector mosquitoes. On the other hand, according Region on the Verge of Malaria Elimination. to the results, the molecular LAMP method is Journal of Kerman University of Medical very useful in detecting infections in the field Sciences. 2020;27(1):36-48. due to its high sensitivity, short test time, and 6. Soleimani-Ahmadi M, Vatandoost H, Zare M, the need for inexpensive equipment. According Turki H, Alizadeh A. Topographical to the Kappa’s measure of agreement, both distribution of anopheline mosquitoes in an methods are fully consistent with each other area under elimination programme in the south of Iran. Malaria journal. 2015;14(1):262. (К=1). Thus, molecular epidemiological studies

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