J Arthropod-Borne Dis, December 2020, 14(4): 353–362 FF Ouma: Application of High-Resolution … 353 http://jad.tums.ac.ir Published Online: December 31, 2020 Original Article Application of High-Resolution Melting (HRM) Technique towards the Detec- tion of Asymptomatic Malaria in a Malaria Endemic Area of Southeastern Iran under Elimination Program Francan Felix Ouma1; *Mehdi Nateghpour1; Afsaneh Motevalli Haghi1; Mehdi Mohebali 1,2; Leila Farivar1; Ahmad Hosseini-Safa1; Sayed Hussain Mosawi3 1Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Center for Research of Endemic Parasites of Iran, Tehran University of Medical Sciences, Tehran, Iran 3Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom, Iran *Corresponding author: Dr Mehdi Nateghpour, E-mail: nateghpourm@sina.tums.ac.ir (Received 30 Sep 2018; accepted 03 Dec 2020) Abstract Background: Asymptomatic malaria, which usually exists in low parasitemia, acts as the Plasmodium species reser- voirs contributing towards malaria transmission. This situation hinders malaria elimination programs in endemic areas, thus necessitating an active case detection with a high sensitive method and treatment of cases. This is why we used a High Resolution Melting (HRM) assay to monitor the trend of asymptomatic malaria in a malaria endemic area of Iran which is under elimination program. Methods: The peripheral blood was sampled from 271 clinically approved non-febrile individuals from a malaria en- demic zone of southeastern Iran for asymptomatic malaria prevalence detection by microscopy, Rapid Diagnostic Tests (RDTs) and HRM methods. The HRM assay was done based on the amplification of 18S SSU rRNA gene. Results: The HRM assay revealed infections from three individuals out of 271 (1.1% asymptomatic malaria prevalence) from the participants, two Iranian natives with Plasmodium vivax infection and one Pakistani immigrant with P. falcipa- rum infection. Neither microscopy nor RDTs detected Plasmodium spp infections from the 271 non-febrile individuals. The nucleotide sequencing analysis of the positive controls used in this study showed a close homology with the refer- ence gene bank sequences of P. falciparum 3D7 (CPO16995.1) and P. vivax Sal-1(UO3079.1). Conclusion: This study revealed a low frequency of asymptomatic malaria trend within malaria endemic areas of southeastern Iran which are under intense elimination program and also the ability of HRM assay in detecting low Plasmodium spp parasitemia beyond the limits of microscopy and RDTs. Keywords: Asymptomatic malaria; High resolution melting (HRM); Microscopy; Rapid diagnostic test (RDTs); Iran Introduction Malaria remains an infection of global con- cern with almost half of the world’s popula- tion being at risk (1, 2). The disease burden re- mains high, in spite of 21% and 29% reported reduction in prevalence and deaths respec- tively between 2010 and 2015 (3, 4). The esti- mated global malaria cases and deaths in 2018 stood at 228 million people and 405,000 peo- ple respectively. Malaria annual global funding estimate stands at US Dollars 2.7 Billion (4). Iran, which is in malaria elimination phase, reports a low malaria prevalence (of 0.87%) with the most endemic areas (including Sistan and Baluchestan, Kerman and Hormozgan Prov- inces) being in the south and southeastern part of the country where most cases are imported from malaria endemic neighboring countries of Afghanistan and Pakistan (5). The continuous exposure of human to ma- laria parasites in malaria endemic areas may create asymptomatic carriers which act as par- asite reservoir harboring gametocyte and con- tribute towards malaria transmission (6). A se- vere malaria disease in these areas occurs most- Copyright © 2020 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, December 2020, 14(4): 353–362 FF Ouma: Application of High-Resolution … 354 http://jad.tums.ac.ir Published Online: December 31, 2020 ly in non-immune individuals such as children or visitors. Asymptomatic malaria is the ma- laria parasitemia of any density without any ordinary symptom in an individual who has not received any recent antimalarial treatment. Asymptomatic malaria results from a partial im- munity which only arrests but does not elimi- nate the infection (7). The asymptomatic ma- laria cases were formerly common in high trans- mission settings in Sub Saharan Africa and some regions of Southeastern Asia but currently have also been reported in low transmission settings of South America as well (8). These infections may be associated with chronic and debilitating episodes of symptomatic parasitem- ia. Early detection and treatments of these cases is of paramount importance towards successful malaria elimination in endemic areas. Plasmo- dium spp. parasitemia involved in asymptomatic malaria infection is usually low and would thus require a high sensitive diagnostic tool (7, 6). Microscopy and Rapid Diagnostic Tests (RDTs) which are the commonly used methods for malaria diagnosis because of their simplicity and inexpensiveness, have a challenge of low sensitivity (9-12). Molecular based techniques can overcome this challenge by not only being able to detect Plasmodium spp. in lower den- sities but are also capable of reliably identify- ing and differentiating between Plasmodium spp and mixed infections as well (10, 11, 13, 14). High resolution Melting (HRM) assay is a post PCR molecular method of genotyping which is attached to PCR amplification. HRM assay is a high sensitive, simple, rapid and accurate method based on the generation of melt curves and is very instrumental towards diagnosing Plasmodium spp. in low parasitemia based on a previously conducted study (10). The aim of this study was to detect Plas- modium spp infections among non-febrile in- dividuals living in a malaria endemic area of Southeastern Iran which is under intense elim- ination exercise using Real Time Polymerase Chain Reaction coupled with High Resolution Melting (Real Time PCR-HRM) analysis. Materials and Methods The study settings This cross sectional study was conducted from 2016 to 2017. The study involved sample collection from Sistan and Baluchestan Prov- ince in Southeastern Iran and laboratory ex- perimental implementation in Malaria Refer- ral Laboratory of Tehran University of Medi- cal Sciences. Ethical consideration The study was approved by the ethical com- mittee of Tehran University of Medical Scienc- es (approval ref. No: IR.TUMS.VCR.REC. 1396.3314). The importance of the study was explained to the participants in their local lan- guage. Consent was obtained from all of the study participants. The Parents and the guard- ians consented on behalf of the study partici- pants with age below 18 years. Study sites This study was conducted in Sarbaz and Chabahar Districts of Sistan and Baluchestan Province in southeast of Iran (Fig. 1). This province is the largest in Iran with an area of 181,785km2 and a population of 2.775 mil- lion. The East of the Province borders malaria endemic countries of Afghanistan and Pakistan and Oman Sea to its South. The province ac- counts for one of the driest regions of Iran with a slight increase in rainfall from east to west, and an obvious rise in humidity in the coastal regions. It has hot and dry weather with about 65mm rainfall annually and accounts for the most malaria cases in Iran. About 92% of ma- laria cases in this region are caused by P. vi- vax and the balance by P. falciparum. In the recent years, Anopheles stephensi and An. cu- licifacies s.l. are the most important malaria transmission vectors in this region. The area ex- periences two malaria transmission peaks which are spring and autumn seasons (15, 16). The hospitals in these areas rely majorly on mi- croscopy and RDTs for malaria diagnosis. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 353–362 FF Ouma: Application of High-Resolution … 355 http://jad.tums.ac.ir Published Online: December 31, 2020 Human blood sampling The sample collection was done from 271 healthy individuals (171 native Iranians and 96 immigrants from Pakistan and Afghanistan) who were residing in the study areas for at least 6 months and had no any signs of febrile illness including fever, chills and rigor. The blood sample collection was done with strict adher- ence to the universal precautions for handling and disposal of human blood including: clean- ing and puncturing the middle finger for prep- aration of thick and thin Giemsa stained blood slides according to the World Health Organi- zation (WHO) malaria microscopy standard op- erating procedure (17). One blood spot from every study participant was put into a DNA banking card (Kawsar® Genomics, Iran) for molecular analysis. Another blood spot was put in RDT cassette (Premier Medical, India) and the results read immediately in the field in accordance to the WHO recommendations (18). The samples were then transferred by air to Tehran for molecular analysis. Patients who were perceived to have febrile illnesses resem- bling malaria infection were excluded from this study but were referred to the hospital. Microscopy examination Microscopic examination was done with strict adherence to World Health Organization (WHO) malaria microscopy standard operating proce- dure (17). The examination was done by two experienced microscopy experts who checked each duplicate of samples from each and eve- ry individual using X100 power objective pow- er ocular lens. The slides were labelled nega- tive if there were no any stage of Plasmodium spp seen after 100 microscopic fields were checked (17). Molecular Analysis Primer selection A pair of primers sequence used in this work was selected based on a previous study by Chua et al. 2016 and was aimed at amplifying the target sequence located at 18s SSU rRNA gene of Plasmodium spp. The forward primer was 5’-GRAACTSSSAACGGCTCATT-3’ and re- verse primer 5’ACTCGATTGATACACACTA- 3’ (10). DNA Extraction DNA extraction was done based on a pre- vious study by Bereczky using Tris-EDTA buff- er (19) Tris-EDTA buffer contains 10mM Tris which is a mixture of Tris base and Tris HCl to a pH of 8.0; and distilled water dissolved 0.1mM EDTA. After the preparation, the Tris EDTA buffer stock solution was kept at a room temperature. Punches from a DNA banking cards were cut into smaller pieces and put in a specific Eppendorf tube. 65µl of TE buffer was added to the Eppendorf tube containing the sample and vortexed. The samples were then put in a water bath at 50 oC for 15 minutes accompa- nied by gentle pressing with a fresh micropi- pette tips for every sample. The water bath temperature was adjusted to 95 oC with 15 minutes heating to elute the DNA from the samples. The samples were then centrifuged at 13,000 RPM for 1 minute to condense the liq- uid on the lid and the wall of the tubes. To confirm the concentration of the DNA, spec- trophotometry (Nanodrop 2000®, Wilmington USA) was performed. The DNA extracts were then stored at -20 oC until was required to be used for HRM analysis (19). The control templates The positive controls were laboratory sam- ples from P. falciparum and P. vivax infected human patients. These patients tested positive for both the microscopy and RDTs. For fur- ther scrutiny of the positive control, 30µl of their DNA templates was sent for sequencing (Macrogen, Korea). The nucleotide sequence was then compared with the sequences in the gene bank. The negative control was distilled water. Reaction Mixture preparation The HRM reaction mixture was made in a final volume of 20µl. The components includ- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 353–362 FF Ouma: Application of High-Resolution … 356 http://jad.tums.ac.ir Published Online: December 31, 2020 ed: 9µl of distilled water, 0.5µl of the forward primer (5’-GRAACTSSSAACGGCTCATT- 3’), 0.5µl of the reverse primer (5’-ACTCGA TTGATACACACTA-3’), 5µl of EvaGreen® dye and 5µl of DNA template. The final vol- ume of each sample was put in HRM strips and fixed with optical caps (Gunster Biotech, Taiwan) and loaded into the HRM machine (Step One plus TM) for analysis. High Resolution Melting (HRM) analysis The HRM analysis set conditions included primary denaturation at 95 oC for 5 minutes, followed by 50 cycles each of: denaturation at 95 oC for 25 seconds, annealing at 55 oC for 30 seconds, extension at 72 oC for 30 seconds and final extension at 72 oC for 5 minutes. The HRM section included temperature increase from 65 oC to 90 oC with a temperature shift interval of 0.3 oC. To check for the consistency of the result, the positive clinical samples were repeated 4 times in different times but under the same con- dition. The HRM curves were generated and analyzed using High Resolution Melt Software v 3.0.1 (step one plus). Results Characteristics of study participants The study participants were residents of Sistan and Baluchestan Province, southeastern of Iran. 64% (n= 175) of the study participants were Iranians and the balance (n= 96) were im- migrants from Afghanistan and Pakistan. All of the study participants had no clinical symp- toms of malaria or any other febrile disease that seems to resemble malaria paroxysm including fever, chills and rigor. 140 of the study partici- pants were males and the rest (131) were females. About 46.4% of the males (n= 65) were native Iranians and the rest (n= 75) were immigrants. About 83.9% of the females (n= 110) were native Iranians and the balance (n= 21) were immigrants. None of the participants had either a history of malaria or has been on an antimalarial therapy within the past six months. Sequence analysis for the positive control samples A multiple sequence alignment between the nucleotide sequences of experimental positive controls of P. falciparum and P. vivax (Fig. 2) and the nucleotide sequences from the gene bank standards of Plasmodium falciparum 3D7 (CPO16995.1) and Plasmodium vivax sal-1 (UO3079.1) done using clustal W2 confirmed a close homology (99.44% and 90.37% respec- tively) between them as shown in the percent- age identity matrix. The phylogenetic tree al- so confirmed a close relation (Fig. 3). Plasmodium spp prevalence detection using the Rapid diagnostic tests (RDTs), Microscopy and High Resolution Melting analysis Microscopy (through checking of 100 fields in thin blood films) and RDTs did not detect Plasmodium infection from 271 clinical sam- ples (Table 1). The NanoDrop carried out con- firmed a good concentration of DNA. The HRM revealed three infections from 271 clinical sam- ples: two P. vivax infections and one P. falci- parum infection. High Resolution Melting analysis The HRM analysis was done using HRM melt software v 3.0.1 (step one plus) (Fig. 4) Various forms of curves were generated in- cluding Amplification plots, derivative melt curves, and aligned melt curves for the three clinical positive Plasmodium spp. based on the two positive controls. The positive clinical samples were easily identified based on the nature of the curves that aligned with the pos- itive control curves. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 353–362 FF Ouma: Application of High-Resolution … 357 http://jad.tums.ac.ir Published Online: December 31, 2020 Fig. 1. Study area in Sistan and Baluchestan Province, Iran, (http://ircmj.com/168.fulltext) Fig. 2. The nucleotide sequence for the experimental positive controls (A) Plasmodium falciparum (MG551534) and (B) P. vivax (MG551535) Fig. 3. The homology between the Plasmodium falciparum and P. vivax experimental positive control samples and the P. falciparum 3D7 (CPO16995.1) and P. vivax sal-1 (UO3079.1) gene bank standards nucleotide sequences. (A) Per- centage identity matrix and (B) phylogenetic tree. Both the identity matrix and the phylogenetic tree shows a close iden- tity between the experimental positive controls and the standards from the gene bank http://jad.tums.ac.ir/ http://ircmj.com/168.fulltext J Arthropod-Borne Dis, December 2020, 14(4): 353–362 FF Ouma: Application of High-Resolution … 358 http://jad.tums.ac.ir Published Online: December 31, 2020 Fig. 4. Melt curves of the positive clinical samples and the experimental positive controls (A) the amplification plot, (B) derivative melt curves and (C) aligned melt curve http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 353–362 FF Ouma: Application of High-Resolution … 359 http://jad.tums.ac.ir Published Online: December 31, 2020 Table 1. Asymptomatic malaria cases detection results based on Rapid diagnostic tests (RDTs), Microscopy and High Resolution Melting assays Nationality Number of Study Participants Number of Microscopy Positive Cases Number of RDTs Positive cases Number of HRM Positive Cases Iranian 175 0 0 Male 1 Female 1 Immigrants 96 0 0 Male 1 Female 0 Total 271 0 0 3 Discussion The curves for the three clinical Plasmodium spp positive samples aligned to the curves of the positive control samples consistently throughout the four trials. 18S SSU rRNA was selected for use in this study based on the previously molecular studies. This region is much conserved and highly variable having multiple copies which are scattered throughout the genome of Plasmodium spp making it suitable for molecular amplification (10, 20). Upon the sequencing of the positive con- trol samples for this study, the nucleotide se- quence of the positive controls used (P. falci- parum and P. vivax) showed a close homol- ogy with the gene bank reference sequences for P. falciparum 3D7 (CPO16995.1) and P. vivax Sal-1(UO3079.1). The principals behind HRM analysis to- wards detections and differentiation of Plas- modium spp is based on the nature of melt curves and melting temperature (Tm). The HRM as- say set up is the same as with the RT-PCR procedure with a difference of incorporation of a fluorescent intercalating dye in HRM that is used to monitor the transition of DNA from double stranded (dsDNA) to single stranded (ssDNA) and assigning the Tm in the process. Tm is a point in which dsDNA is equal to ssDNA, this point is very significant towards species differentiation and Plasmodium spp mixed infections detection (21, 22). In this study, three individuals out of 271 non-febrile study participants (1.1 %) from the malaria zone of Iran have been found to be infected with Plasmodium spp. This is in a close conformity with other molecular based asymptomatic malaria surveillance findings from the same malaria endemic areas of Iran: Amirshakeri et al. and also Turki et al. report- ed 0.33% and 1.5% asymptomatic malaria in- fections respectively from the surveys they con- ducted (22, 23). The parasitemia involved in these infections could neither be detected by microscopy nor RDTs. However in another mi- croscopy based asymptomatic malaria preva- lence survey that has been conducted by Nateghpour et al. in Iranshahr District of Sis- tan and Baluchestan Province, neighboring the studied districts, they reported 1.6% and 0.6% Plasmodium spp prevalence among Afghani- stan immigrants and native Iranians respec- tively (24). Obviously, according to WHO guide- lines the number of parasites per microliter of blood is crucial for detecting them either with microscope or RDTs. The density of parasite is not less than ten and forty for microscopy and RDTs respectively. So, discrepancies among results of the mentioned references may be in- terpreted due to the above mentioned reason. These infections if left undetected and un- treated would act as reservoirs and play a cru- cial role towards the transmission of malaria within a population where they are prevalent and would thus consequently delay or prevent malaria control and elimination exercises in ma- laria endemic areas. On the other hand, some http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 353–362 FF Ouma: Application of High-Resolution … 360 http://jad.tums.ac.ir Published Online: December 31, 2020 molecular based studies conducted in the south- eastern malarious areas of Iran resulted in ab- sence of asymptomatic malaria (25, 26). Kipanga and colleagues were able to diag- nose and detect Plasmodium spp from micro- scopically negative febrile patients by using PCR coupled with HRM from hospitals in Ken- ya who could not be put on antimalarial drugs (12). Chua et al. identified Plasmodium spp in 11 out of 14 microscopically negative febrile patients using RT-PCR coupled with HRM (10). Iran has achieved tremendous successes to- wards the elimination of malaria by using meth- ods like: passive and active case detection, vec- tor control methods, and effective treatment using artemisinin combination therapy (ACT). These strategies have reduced malaria infec- tions in the country from 12,294 to 1,251 be- tween 2000 and 2014 (22-24). Asymptomatic malaria may however rekindle malaria infec- tions or slow down the pace of the elimination programs being undertaken. Apart from the aforesaid, HRM assay has also been successfully used in other parasito- logical studies including: identification of mu- tant strains of P. falciparum that are drug re- sistant; diagnosing the genetic diversity of Di- entamoeba fragilis isolates of irritable bowel syndrome patients, analyzing the single nucle- otide polymorphism of Echinococcus granu- losus, detection and identification of Old World Leishmania, detection and identification of Human Hookworm infections. These studies have demonstrated a high sensitivity nature of HRM in diagnosis compared to other parasito- logical methods (27-31). Although HRM as- say is very sensitive in diagnostics, its initial cost and operational costs are more expensive than microscopy and RDTs. Conclusion In conclusion HRM assay is a favorable method towards the rapid detection of malaria in low parasitemia below the LoD of micros- copy and RDTs and it is therefore important in areas where malaria elimination exercises are being carried out particularly if the malaria pol- icy makers want to remove most hidden ob- stacles including asymptomatic malaria from their pathway. This method is also capable of differentiating between Plasmodium spp. by just using a single pair of primer sequence mak- ing it simpler to use compared to other diag- nostic methods. Coming up with this kind of an assay which would incorporate DNA ex- traction from the samples prior to their ampli- fications would be a great development as it would avoid the errors associated with the DNA extractions that may affect the results of HRM assay. 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