INTRODUCTION:

Among the four species of genus plasmodium, Plasmodium vivax is the second most prevalent one that cause malaria^[1]. Several biologic factors make the elimination and control of Plasmodium vivax so hard, such as its low density in blood which makes its detection difficult, presence of resistant hepatic forms which cause relapse or emergence of early sexual stage of the parasite, which leads to easy transmission of the parasite^[1]. The rate of P. vivax incidence tends to be higher than P. falciparum among younger age patients when both of the parasites are prevalent because each inoculation of P. vivax may be followed by many relapses^[2]. It was shown that the nested PCR amplification of the sequences of ribosomal RNA genes is the most validated and used molecular diagnostic techniques. The term (nested) indicates an initial amplification by applying the genus-wide sequences primers followed by species specific primer to facilitate the definitive diagnostic step^[2].

The use of ELISA technique which depend completely on semi-purified antigens is very rare due to difficult obtaining of P. vivax in vitro. According to this restriction, we examined the recombinant proteins which represent 19 kDa C-terminal region of the surface protein-1 of P. vivax merozoite (MSP119), which might be a useful serological method for the detection of malarial infection^[3]. The policy of donor deferral in different countries restrict blood donation of people who recently emigrated or travelled from endemic areas or who have recent clinical malaria infection. Recent studies revealed the incidence of Plasmodium knowlesi, a pathogenic species of Simian origin, in populations of Southeast Asia^[4,5]. At present, the commercial ELISA is being used in France, Austuralia and the United Kingdom, and the discussion of of questionnaire-deferred donors is being reinstated in the United States and Canada^[6,7]. MSP1-P19 antigens of Plasmodium ovale and Plasmodium malariae were effectively applied to detect the antibodies of these two parasites in human IFA- and microscopy-confirmed methods. The detection of IgM and IgG antibodies in experimentally-infected chimpanzees demonstrated a further evidence of serological testing efficacy. In addition, it was shown that combining of MSP1-p19 antigens of the four human infective species of plasmodium into a single prototype antibody test is feasible^[8].

MATERIAL AND METHODS:

In the current study, blood samples were collected from 100 Iraqi malaria patients infected with Plasmodium Vivax during the period from 1^st may 2018 to 1^st April 2019, The sero-diagnosis was done by detection of malarial antigens of panel detection score (PDS) of the corresponding Plasmodium species at 200 parasites per μL of blood. The genetic analysis was determined by amplification of Plasmodium vivax k13 gene by using the nested PCR technique, and the k13 gene primer was designed as explained before. Forward and reverse primers of the first PCR amplification round were (5′-CGGAGTGACCAAATCTGGGA-3′) and (5′-GGGAATCTGGTGGTAACAGC-3′) respectively. The region in chromosome 13 sequence of P. vivax and the size of the product size was expected to be approx. 849 bp. Both the first and second PCR reactions contained 2.6µL DNA templates, but when the second reaction was the product, the first reaction was used as a template. 14µL of 2× Taq PCR mixed system (containing Taq enzyme) has been provided by Qiagen Biotech company, 0.7µL each forward and reverse primers (20µmol/L), and ddH₂O for a volume of 25µL.

Statistical Analysis:

Data analysis was done by using the SPSS Vr.24. t – test and Monte Carlo test (MCP) were applied at 5% & 1% levels of significance.

RESULTS:

The panel detection score (PDS) range for the corresponding Plasmodium vivax at 200 parasites per μL of blood extracted from plasmodium Vivax antigen by using Pv-pLDH, Pvom-pLDH and aldolase antigens was shown in table (1).

Figure 1: Detection of the DNA of (k13 gene) genes Lane M markers corresponding to 500 bp ladder (fermintus), lanes 1 & 2and 3 the k13 gene bands with 849 bp.

Table 1: The range of panel detection scores of quality controll RDTs product testing.

Species	Antigen	PDS^a range
P. vivax	Pv-pLDH	51% (0%-100%)
P. vivax	Pvom-pLDH^b	75% (63%-91%)
P. vivax	aldolase	34% (0%-82%)

The correlation between gene mutants and Bulaquine drug resistance:

Blood specimens of the 100 P.Vivax malaria Iraqi patients, who finished in vivo Bulaquine resistance testing were paired for the k13 gene polymorphism analysis.

Consequently, among the 8 cases, only 5 mutation types were detected in k13) gene, F446I, C469Y, A676D, N458Y and P574L types. Table 2 illustrated mutations at loci of k13 gene:

Table 2: Correlation between gene mutant and Bulaquine drug resistance.

Mutation Loci	Frequency	OR value
C469Y A676D, N458Y, P574L	28	1.412
F446I	23	1.284

Change of k13 gene sequences:

The DNA sequence of the (k13) genes obtained from 8 P.Vivax malaria Bulaquine resistance cases in Iraqi patients showed mutation in G449G, T451T,G674G, F446I and G450V, and the mutations were GGT to GGG, TTT to TTC, GGG to GGA, TTT to GTA and GGA to GTA respectively as shown in (Table 3).

Table 3: Genetic sequences in (k13) gene of P. Vivax among Iraqi patients

Mutation position	Nucleotide Chang	Change	Code
G449G	1347 T > G	GGT > GGG	1,0.3
T451T	1353 T > C	TTT > TTC	1,0.3
G674G	2022 G > A	GGG > GGA	1,0.3
F4461	1338 T > A	TTT > ATT	130,33.6
G450V	1349 G > T	GGA > GTA	1,0.3

DISCUSSION:

Plasmodium Vivax is one of mlarial parasite, which causes benign haemorrhagic fever, there is a wide spread of malaria in Iraq, especially in the northern regions of Iraq^[9]. Malarial parasites work to make many genetic mutations and in several sites, on the chromosomes, to be resistant against several treatments, which agreed with^[10] who found that the antimalarial drugs including folate synthesis in the cytosol have targeted parasite-specific biological processes, which are necessary for protein and pyrimidine biosynthesis in the apicoplast of the antimalarial drugs and the genetic change in the loci number may cause the drug resistance. The hemoglobin variant range, apart from Hemoglobin S, emerged from evolutionary selection by the parasite, included other Hb gene variants such as (Hb-C) and (Hb-E) and the regulatory (Hb-A) and (Hb-B) defects, which lead to the occurrence of (α) and (β) thalassemias^[11]. In a study, it was found that (159) gene amplifications and (148) nonsynonymous alterations in (83) genes were related to drug resisting acquisition, as gene amplification participated in about 1/3 of drug the resistance acquisitions^[12]. The alteration of the malaria parasite's genetic sequence renders it more virulent and more resistant to traditional drugs^[13]. The area in antimalarial drug markets has been partially filled by phenotypic screens. However, new treatments will only be provided by direct development of drugs depending on the understanding of the fundamental biology of the parasite itself. This study focused on the abnormal cell cycle of the genus Plasmodium, which may provide a good source of drug targets in addition to a subject regarding the essential biological interests ^[14]. Sequences of DNA on the (k13) gene taken from 8 cases of Vivax malaria Bulaquine drug resistance in Iraqi patients indicated that the mutation in G449G, T451T, G674G, F446I and G450V were GGT to GGG, TTT to TTC, GGG to GGA, TTT to GTA and GGA to GTA respectively. This genetic alteration has really changed the normal characteristics of malaria parasite, making it resist the treatments given to malaria patients, which targets the sites of protein synthesis or cellular division of malaria, and these findimgs matched with^[10] who observed the appearance of parasites with low sensitivity to artemisinin derivatives and ACT partner drugs, leading to high treatment failure rates. Here we have shown the recent progresses to understand the way of antimalarial drug action and resistance development, and we discuss using new strategies to combat treatment resistant effectively ^[15].

CONCLUSION:

1. Five mutation types were detected in k13) gene, F446I, C469Y, A676D, N458Y and P574L types.

2. Eight P. Vivax malaria Bulaquine resistance cases in Iraqi patients showed mutation in G449G, T451T, G674G, F446I and G450V,

3. The mutations were GGT to GGG, TTT to TTC, GGG to GGA, TTT to GTA and GGA to GTA respectively.

REFERENCES:

1. Ding, X. C., Ade, M. P. and Baird, J. K. et al., Defining the next generation of Plasmodium vivax diagnostic tests for control and elimination: Target product profiles, PLoS Negl Trop Dis. 2017 Apr; 11(4): e0005516.

2. Baird, J. K. Valecha, N. and Duparc, S. et al, Diagnosis and Treatment of Plasmodium vivax Malaria, Am J Trop Med Hyg. 2016 Dec 28; 95 (6 Suppl): 35–51.

3. Rodrigues MH, Cunha MG, and Machado RL, et al, Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1. Malar J. 2003 Nov 14;2(1):39.

4. Cox-Singh, J., T. M. Davis, K. S. Lee, S. S. Shamsul, A. Matusop, S. Ratnam, H. A. Rahman, D. J. Conway, and B. Singh. 2008. Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening. Clin. Infect. Dis. 46:165-171.

5. Cox-Singh, J., and B. Singh. 2008. Knowlesi malaria: newly emergent and of public health importance? Trends Parasitol. 24:406-410.

6. Elghouzzi, M. H., A. Senegas, T. Steinmetz, P. Guntz, V. Barlet, A. Assal, P. Gallian, P. Volle, C. Chuteau, M. Beolet, S. Berrebi, D. Filisetti, C. Doderer, T. Abdelrahman, and E. Candolfi. 2008. Multicentric evaluation of the DiaMed enzyme-linked immunosorbent assay malaria antibody test for screening of blood donors for malaria. Vox Sang. 94:33-40.

7. Kitchen, A. D., and P. L. Chiodini. 2006. Malaria and blood transfusion. Vox Sang. 90:77-84.

8. Muerhoff, A. S. Birkenmeyer, L. G. and Coffey, R. et al, Detection of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae Merozoite Surface Protein 1-p19 Antibodies in Human Malaria Patients and Experimentally Infected Nonhuman Primates, DOI: 10.1128/CVI.00196-10:2017.

9. Mark D. Gershman, Emily S. and Jentes, Rhett J.et al, Yellow Fever Vaccine & Malaria Prophylaxis information by country, Infectious Diseases Related to Travel, CDC 2019.

10. Blasco, B. Leroy, D. and Fidock, D. A., Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic, Nat Med. 2017 4; 23(8): 917–928.

11. Dominic P. Kwiatkowski, How Malaria Has Affected the Human Genome and What Human Genetics Can Teach Us about Malaria, Am J Hum Genet. 2005 Aug; 77(2): 171–192.

12. Cowell, A. N. Istvan, E. S. and Lukens, A. K. et al, Mapping the malaria parasite druggable genome by using in vitro evolution and chemogenomics, science Vol. 359, Issue 6372, pp. 191-199.

13. Cowell, A. and Winzeler, E. Exploration of the Plasmodium falciparum Resistome and Druggable Genome Reveals New Mechanisms of Drug Resistance and Antimalarial Targets, Microbiol Insights. 2018; 11: 1178636118808529.

14. Matthews, H. Duffy, C. W., and Merrick, C. J. Checks and balances? DNA replication and the cell cycle in Plasmodium, Parasit Vectors. 2018; 11: 216.

15. Blasco, B Leroy, D. and Fidock D. A. Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic, Nat Med. 2017 4; 23(8): 917–928.

Received on 05.07.2019 Modified on 10.08.2019

Research J. Pharm. and Tech. 2020; 13(1): 339-341.

DOI: 10.5958/0974-360X.2020.00068.2