J Arthropod-Borne Dis, June 2019, 13(2): 206–213 BH Habibi-Shorkaei et al.: Identification of … 206 http://jad.tums.ac.ir Published Online: June 24, 2019 Original Article Identification of Alleles in the MSP1 Gene Related to Complicated Malaria in Patients Infected with Plasmodium falciparum in Southeast of Iran Bentol Hoda Habibi-Shorkaei1; Afsaneh Motevalli-Haghi1; *Mehdi Nateghpour1, 2; Leila Farivar1; Homa Hajjaran1; Soudabeh Etemadi1 1Department of 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 (Received 20 Jun 2017; accepted 12 Jan 2019) Abstract Background: To overcome human malaria problem several solutions have been employed including extensive studies in the field of Plasmodia relevant antigens. The aim of this study was to determine allelic variation in the MSP1 gene of Plasmodium falciparum among some falciparum malaria-infected patients in Southeastern Iran. Methods: Twenty P. falciparum positive cases were enrolled from Sistan and Baluchistan Province, southeastern Iran in 2013–15. From each case, 1.5ml of peripheral blood was collected into EDTA contained tubes. Thick and thin blood smears were stained with standard Giemsa stain and were checked with conventional microscopical method. DNA was extracted from blood samples and amplification of block 2 MSP1 was performed using specific primers. Gel electropho- resis was done and results showed some amplification fragments corresponding to block 2 regions of Pf MSP1 gene. Finally, four samples from different allelic types were sent for sequencing process. Results: Fragments were different in size, so classified into six allelic types as kinds of 1–6 based on happening fre- quencies. Digestion of PCR products revealed two sub allelic types (A and B) within allelic types 2 and 3, but not in al- lelic types 1, 4, 5 and 6. Twenty percent of samples were sent for sequencing. Sequence alignment showed 78.95% to 91.83% identity between samples. Conclusion: Identity between samples and phylogenetic tree revealed that there is an extensive diversity range among isolates. Fifty percent of the isolates were under the risk of complicated malaria. Two of these patients (10%) needed special care and recovery was obtained after getting hospital services. Keywords: Plasmodium falciparum; Sever malaria; Iran; PCR-RFLP Introduction Malaria is an ancient problem created many calamities against human for many centuries. Parasites of the infection cause illness to 216 million persons and almost 445000 deaths per year. Pregnant women and children under 5yr old are two greatest groups damaged from this infection (1–3). The fearful condition of the dis- ease in human is caused by Plasmodium fal- ciparum. Cerebral malaria, blackwater fever, severe anemia and some other disorders occur in individuals who infected with P. falciparum. Such problems can result in death among the infected people if is not being treated with suit- able drug. To combat these conditions several malar- ia vaccines were produced and tested amongst some volunteer in clinical trials, but with poor consequences due to some reasons including gene diversities phenomenon in P. falciparum isolates. However, diversities in MSP1 gene demonstrate different alleles which such diver- sities can reveal different aspects of phenom- ena in P. falciparum. One of the most important aspects of the phenomena is emergence a num- ber of complications in patients who infected with P. falciparum (4, 5). Presence of some MSP1 sub allelic types have been seen in those individuals who infect- *Corresponding author: Dr Mehdi Nateghpour, E-mail: nateghpourm@tums.ac.ir J Arthropod-Borne Dis, June 2019, 13(2): 206–213 BH Habibi-Shorkaei et al.: Identification of … 207 http://jad.tums.ac.ir Published Online: June 24, 2019 ed with cerebral malaria. Studies of genetic di- versity can provide valuable data in different aspects of P. falciparum malaria such as fore- casting complicated cases in the infection. Plas- modium falciparum MSP1 gene produces a surface protein in merozoites in blood schizog- ony phase and it is a major candidate for provid- ing a vaccine (6), but its notable diversity in dif- ferent isolates is an obstacle within the pro- gress. The gene produces a 190kDa glycopro- tein including seven variable blocks putting at different places among five conserved and five semi-conserved blocks (5). Diversity recogni- tion of the gene is important to determine al- leles resulting severe malaria. The aim of this study was to determine al- lelic variation in the MSP1 gene of P. falcipa- rum among some malaria patients living in ma- larious areas of Sistan and Baluchistan Prov- ince of Iran in view of presence or absence of MSP1 alleles that cause cerebral malaria, us- ing PCR-RFLP technique. Materials and Methods Study area Sistan and Baluchistan Province is located in southeastern Iran. The latitude and longi- tude of the province are 27º0′ N and 62º0′ E respectively. The weather is warm and low hu- mid in north of the province with average tem- perature of 31 ºC, and warm and humid in south with average temperature of 30.2 ºC. Plasmo- dium vivax and P. falciparum are prevalent Plasmodia species in the studied area. Moreo- ver, Anopheles culicifacies and An. stephensi are the main vectors in southeastern Iran. The province is located on neighboring Pakistan and southwestern part of Afghanistan with 978 and 300km joint borderline respectively. Immigra- tion load of Afghani and Pakistani population in the province is high with notable imported malaria infection. All the samples for this study were collected from those individuals who were living for long time in the studied areas. Blood samples Twenty P. falciparum positive cases were enrolled between 2013 and 2015 for this study. From each case, 1.5ml of peripheral blood was collected into EDTA contained tubes. Previous to sampling an informed consent was prepared of the registered individuals. Detection of parasites Thick and thin blood smears were stained with standard Giemsa stain and the slides were checked with conventional microscopical meth- od with two expert microscopists. Mix infections were taken out from the study. Samples were stored at -20 ºC until DNA extraction processes. DNA Extraction DNA of P. falciparum was extracted from blood samples by using a high pure PCR template preparation kit (Roch REF: 11796828001) ac- cording to the manufacturer instructions. PCR-RFLP PCR amplification of the block2 MSP1 was performed in 25µl reaction that containing, 10 µl Distilled water, 10µl master mix, 3ng DNA, 1µl forward primer, 1µl revers primer. Designing primers and PCR condition were performed (7). The sets of following primers were used for amplification: Forward 5′CTAGAAGCTTTA GAAGATGCAGTATTG3′ Revers 5′GTACG TCTAATTCATTTGCACG AAT3′ PCR am- plification was carried out using initial dena- turation about 5min in 95 ºC, 30 cycles of 1 min at 94 ºC, 2min in 57 ºC, 2min in 72 ºC and final extension 5min in 72 ºC in a thermal cy- cler. Four µl of PCR product was run on 1.5% agarose gel with simply safe (EUREx, Cat: E 4600-01). Finally the process was visualized with gel duct and resulted in some photographs. RFLP (Restriction Fragment Length Poly- morphism) Product amplification of MSP1 gene block 2 zones was digested with Dra-1 endonuclease with active site of TTT AAA (Cat: FD0224). J Arthropod-Borne Dis, June 2019, 13(2): 206–213 BH Habibi-Shorkaei et al.: Identification of … 208 http://jad.tums.ac.ir Published Online: June 24, 2019 The digestion process was carried out in a 30 µl composition of 17µl distilled water, 2µl 10 X fast digest buffer, 10µl PCR product and 1 µl fast digest enzyme. The compound was in- cubated at 37 ºC for 5min and the incubation was continued at 65 ºC for 5min to inactive the en- zyme. Overall 18µl of the digested PCR prod- uct was analyzed on a 3% agarose gel and then stained with simply safe stain. Visualizing pro- cess was conducted as mentioned previously. Sequencing Four PCR products were selected for se- quence processes and analyzed with ClustalW2 and Blast software. Results Genetic diversity of P. falciparum block2 MSP1 gene was considered from clinical iso- lates collected from Sistan and Baluchistan Prov- ince in southeastern Iran (Table 1). All of the 20 isolates showed gene amplified expression in block 2 regions of MSP1 gene. Variation of amplified products was identified from 480 to 600bp (Fig. 1). The isolates were classified into six allelic types as kinds of 1–6 based on hap- pening frequencies. Type 1 possessed the high- est frequency (50%) and allelic types 4, 5, 6 the lowest frequency (5%) (Table 2). PCR products were digested with Dra-1 re- striction enzyme. RFLP identified the subtypes of allelic types among the isolates. Digestion of PCR products revealed two sub allelic (types A and B) within allelic types 2 and 3. While al- lelic types 1, 4, 5, 6 did not show any subtype (Table 3, Fig. 2). The phylogenetic tree showed a wide diversity among the studied samples which some of them had considerable homol- ogy with 3D7 isolate and some others had more homology with HN2 China isolate. The Belem strain of P. vivax was used as outgroup for this analysis (Table 4). Table 1. Demographic data of patients based on nationality, gender and age Nationality Gender Age (yr) Total Male Female < 20 20–40 > 40 Iranian 10 4 3 9 2 14 Afghan 2 2 1 2 1 4 Pakistani 0 2 1 1 0 2 Total 12 8 5 12 3 20 Table 2. Allelic types of MSP1 (Block2) of Plasmodium falciparum isolates based on molecular weight of PCR prod- ucts (n= 20) Size of PCR product Allelic type % No of P. falciparum isolate Allelic frequency % 500bp 1 10 50 550bp 2 5 25 600bp 3 2 10 480bp 4 1 5 570bp 5 1 5 580bp 6 1 5 J Arthropod-Borne Dis, June 2019, 13(2): 206–213 BH Habibi-Shorkaei et al.: Identification of … 209 http://jad.tums.ac.ir Published Online: June 24, 2019 Fig. 1. PCR product of MSP1 gene (block 2) of Plasmodium falciparum Eight isolates were visualized with 2% agarose gel and stained with simply safe. M= DNA size Marker; lane1–8 isolates, NC= negative control Table 3. Allelic and sub allelic types of MSP1 (block2) of Plasmodium falciparum isolates on the basis of PCR-RFLP (digested with Dra-1 restriction enzyme) (n= 20) Allelic type Size of PCR product No of iso- lates Sub allelic type Size of digested fragment No of isolate Frequency % 1 500 10/20=50% --------- 250,250 10/10 100 2 550 5/20=25% 2A 2B 270,280 250,300 3/5 2/5 60 40 3 600 2/20=10% 3A 3B 280,320 250,350 1/2 1/2 50 50 4 480 1/20=5% -------- 250,230 1/1 100 5 570 1/20=5% -------- 280,290 1/1 100 6 580 1/20=5% -------- 280,300 1/1 100 Table 4. Percent Identity among isolates collected from Iran (Accession number: KY026097.1, KYO26098.1, KY026095.1, KY026096.1) and Plasmodium falciparum 3D7 (Accession number XM_001352134.1) and HN2 isolate from China (Accession number AF062349.1). Plasmodium vivax Belem isolate used an out group 1 2 3 4 5 6 7 vivax 3 D7 If 77a If 140 If 133 If 95a china 100.00 43.69 43.54 43.74 40.41 39.28 41.36 43.69 100.00 90.26 92.20 78.21 79.71 83.63 43.54 90.26 100.00 91.83 78.95 79.15 78.85 43.74 92.20 91.83 100.00 80.38 79.75 79.27 40.41 78.21 78.95 80.38 100.00 90.37 91.10 39.28 79.71 79.15 79.75 90.37 100.00 91.60 41.36 83.63 78.85 79.27 91.10 91.60 100.00 J Arthropod-Borne Dis, June 2019, 13(2): 206–213 BH Habibi-Shorkaei et al.: Identification of … 210 http://jad.tums.ac.ir Published Online: June 24, 2019 Fig. 2. RFLP pattern of MSP1 gene block 2(digested with Dra-1) Plasmodium falciparum isolates. Digested products were resolved on 4% agarose gel. Lane M-DNA size marker, lane1 type 4 allele, lane 2 type 3 allele (3A sub allel), lane 3 type 1 allele, lane 4 type 5 allele, lane 5 type 6 allele, NC: negative control. Fig. 3. Partial nucleotide sequences alignment of P.f. MSP1 gene in Plasmodium falciparum isolated collected from malarious areas of Iran and were compared to the genes of China (HN2), vivax (Belem) and 3D7 reference strains. J Arthropod-Borne Dis, June 2019, 13(2): 206–213 BH Habibi-Shorkaei et al.: Identification of … 211 http://jad.tums.ac.ir Published Online: June 24, 2019 Fig. 4. Phylogenetic tree between isolates collected from southeastern Iran (Accession number: KY026097.1, KYO26098.1, KY026095.1, KY026096.1) and Plasmodium falciparum 3D7 (Accession number XM_001352134.1) and CHINA HN2 (Accession number AF062349.1). Plasmodium vivax Belem isolate used an out group. Discussion Investigations about genetic diversities in parasite population can prepare much infor- mation in the field of geographical distribu- tion, genomic markers, clinical manifestation, drug resistance and gene polymorphism for relevant researchers (8-12). Genetic diversity plays a crucial role in the natural acquisition of immunity in malaria disease and it is a ma- jor problem in the pathway of control strate- gies to prevent the distribution of the infec- tion. Combating drug resistance in malaria parasites or producing potential vaccines re- quire comprehension of population structure of the parasite (13). For this purpose, a lot of studies have been conducted by researchers to access genomic markers and polymorphism of the parasite. Among the studies about diver- sity of PfMSP1 gene, a few of them have been focused on relationship between the gene and probable malignancy of P. falciparum (14). There is a significant correlation between di- versity in MSP1 gene and complicated cases of malaria, and in contrast to them, some oth- ers mentioned there is no association among them (15-18, 7, 10). Obtaining different results about the men- tioned subject are due to the different employed methods to found diversity in this gene. For ex- ample nested PCR was used to found frequen- cy of K1, Mad20 and Ro33 alleles in the para- site population (18-20). However, PCR-RFLP was used to report diversity in the gene and reported five allelic types. Nine different al- leles were reported from of 67 isolates and mentioned MSP1 gene in P. falciparum have shown an extended variation in size and pat- tern (19). Any correlation was mentioned be- tween these alleles and malignancy in P. fal- ciparum. There was no significant relationship between genetic diversity of MSP1 and sever- ity in malaria, however, Mad20 allelic type in- creased with the severity of malaria. On the other hand, there are a few alleles in Pf MSP1 gene which are more predominant in cerebral malaria when they used PCR-RFLP to inves- tigate diversity in the gene. Five allelic types including eight genetic types were identified, they implied that sub allelic types 2A and 3A with 300–200 and 280–200bp were more prev- alence in patients got involved with cerebral ma- laria these sub allelic types were recognized when they used PCR-RFLP with Dra-1 re- striction enzyme. Moreover, sub allelic 2B with 250–25bp was seen in both cerebral and non- cerebral malaria cases. Presence of certain al- leles may cause severe disease in patients. In the present study six allelic types were classified by PCR-RFLP, also two sub allelic types were identified when PCR products di- gested with Dra-1, results showed that there is a large diversity in the weight of the products fragments when compared with Farooq et al results (17). So, diversity of the gene is high in various areas. Anyway, 65% of the patients who registered in the present study showed al- lelic types like reported by Farooq and 50% were under the risk of complicated malaria (17). Two of these patients (10%) needed special care and J Arthropod-Borne Dis, June 2019, 13(2): 206–213 BH Habibi-Shorkaei et al.: Identification of … 212 http://jad.tums.ac.ir Published Online: June 24, 2019 recovery was obtained after getting hospital ser- vices. Isolating the genes that involve in pro- ducing cerebral malaria provides a valuable opportunity for prevention of deleterious compli- cations of falciparum malaria in endemic areas. Moreover, sequence alignment among the samples showed 78.95% to 91.83% identity. There is an extension of diversity among the samples. Phylogenetic tree revealed that there is an extensive diversity range between isolates so that some of the isolates achieved in the pre- sent study showed a close relationship with isolate HN2 from China and the others have this association P. falciparum 3D7. There is high diversity in the gene in the specific pop- ulation of the parasite. 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