DNA Sequence Polymorphism of the Lactate Dehydrogenase Genefrom Iranian Plasmodium vivax and Plasmodium falciparum Isolates.

BACKGROUND: Parasite lactate dehydrogenase (pLDH) is extensively employed as malaria rapid diagnostic tests (RDTs). Moreover, it is a well-known drug target candidate. However, the genetic diversity of this gene might influence performance of RDT kits and its drug target candidacy. This study aimed to determine polymorphism of pLDH gene from Iranian isolates of P. vivax and P. falciparum.
METHODS: Genomic DNA was extracted from whole blood of microscopically confirmed P. vivax and P. falciparum infected patients. pLDH gene of P. falciparum and P. vivax was amplified using conventional PCR from 43 symptomatic malaria patients from Sistan and Baluchistan Province, Southeast Iran from 2012 to 2013.
RESULTS: Sequence analysis of 15 P. vivax LDH showed fourteen had 100% identity with P. vivax Sal-1 and Belem strains. Two nucleotide substitutions were detected with only one resulted in amino acid change. Analysis of P. falciparum LDH sequences showed six of the seven sequences had 100% homology with P. falciparum 3D7 and Mzr-1. Moreover, PfLDH displayed three nucleotide changes that resulted in changing only one amino acid. PvLDH and PfLDH showed 75%-76% nucleotide and 90.4%-90.76% amino acid homology.
CONCLUSION: pLDH gene from Iranian P. falciparum and P. vivax isolates displayed 98.8-100% homology with 1-3 nucleotide substitutions. This indicated this gene was relatively conserved. Additional studies can be done weather this genetic variation can influence the performance of pLDH based RDTs or not.

Introduction

Malaria is one of the most important infectious diseases in the world. Although malaria is preventable and curable, it still causes high morbidity and mortality (1). According to the recent 2013 WHO report, globally an estimated of 3.4 billon people are at risk of malaria. In this report, WHO estimated 207 million malaria cases and 627,000 deaths occurred globally in 2012 (2). The majority of the global burden of human malaria is caused by Plasmodium falciparum and P. vivax (3). P. falciparum is the most deadly Plasmodium species responsible for about 90% of malaria deaths, mainly in Africa (4) and P. vivax is the most cause of malaria infection in the world (1). P. vivax is accountable for 25–40% of the annual bouts of malaria worldwide (4). In Iran, 2,714,648 individuals (4% of the total population) mainly living in southern provinces namely Sistan and Baluchistan, Kerman and Hormozgan are at risk of malaria (5). P. vivax is the most prevalent species reported among the malaria patients in Iran annually (6). However, a considerable decrease of malaria cases has been reported within the past few years in Iran. Since malaria elimination program has commence from a few years ago in the country (7), for steady continuation of the program rapid and accurate diagnosis of malaria parasites play an important role in opportune case finding and treatment which result in on time control and elimination of the infection.

Conventional microscopic examination of Giemsa stained thick and thin blood smears has been accepted as golden standard method for malaria diagnosis up to now. Although malaria microscopy contains some advantages including cost, availability and relative sensitivity (8–10), it bears some disadvantages such as time consuming and labor intensity (9). The WHO has recently reiterated “the urgent need for simple and cost-effective diagnostic tests for malaria to overcome the deficiencies of light microscopy” and clinical diagnosis (10, 11). Based on WHO advise rapid diagnostic tests can be replaced with microscopic method in remote and isolated areas particularly when trained and skilled personnel is not available (12–14). Utilizing parasite lactate dehydrogenase (pLDH) in RDTs has shown better sensitivity for diagnosing low level of parasitemia in comparison with other malaria proteins. Moreover, the amount of pLDH indicates to metabolically presence of P. vivax due to short stability of pLDH in the body (13). pLDH plays role of a coenzyme due to involving the oxidation of lactate to pyruvate with nicotinamide adenine dinucleotide (NAD) (15). Inhibition of the malarial LDH enzyme prevents the production of ATP and results to death of the Plasmodium parasites (13); it becomes an attractive drug target candidate (16).

The genetic diversity of pLDH might influence its drug target candidacy and the sensitivity of RDT kits. As far as we know, until now the genetic variation of pLDH gene in P. vivax and P. falciparum infections were not reported in Iran. This study aimed to detect the polymorphism of pLDH gene from Iranian strains of P. vivax and P. falciparum. Obviously, understanding such polymorphism is important for designing or improving RDT kits. It can also give information about the molecular details of P. falciparum LDH (PfLDH) and P. vivax LDH (PvLDH) genes for designing a new drug.

Materials and Methods

Totally 43 whole blood samples were collected from P. vivax and P. falciparum infected patients in Sistan and Baluchestan Province located in southeast of Iran from 2012 to 2013. Sistan and Baluchestan Province is bordered with Afghanistan and Pakistan to the east and Oman Sea in south. It has hot and dry weather with about 65mms rainfall annually. Thirty-three samples for P. vivax and 10 samples for P. falciparum were confirmed positive by light microscopic examination of Giemsa stained thick and thin blood smears. One ml of blood was collected into tubes containing EDTA anticoagulant, placed immediately at −20 °C for further analysis.

This study was approved by Tehran University of Medical Sciences Ethical Committee.

Genomic DNA extraction

DNA was extracted from 200 μl of whole blood samples of 33 P. vivax and 10 P. falciparum malaria infected patients using, ACCU-PreP® kit, Genomic DNA extraction kit (BIONEER, Seoul, Korea) based on the manufacturer instructions. PvLDH and PfLDH genes were amplified and sequenced to analyze the genetic variations.

PCR amplification

Nucleotide sequences corresponding to PvLDH and PfLDH genes were amplified using the following sets of primers using conventional PCR. PvLDH gene amplification was conducted using: Forward: 5′-ATGACGCCGAAACCCAAAAT-3′ and Reverse: 5′-ACCTTTAAATGAGCGCCTTCAT-3′, on the other hand PfLDH gene was also amplified by F: 5′-AGATGGCACCAAAAGCAAAAAT-3′ and R: 5′-ACCTTTAAGCTAATGCCTTCAT-3′. PvLDH primers were designed based on P. vivax Sal-1 (XM_001615570.1) and P. vivax Belem (DQ060151.1) strains from GenBank whereas PfLDH primers designed based on the reference sequence P. falciparum 3D7 (XM_001349953.1) strain in GenBank. DNA was extracted from whole blood of a healthy person living in non-endemic area as a negative control for using in amplification process.

PCR reaction was performed in 25μl reaction volumes containing 1μlof each forward and reverse primers (10 pmol), 10 μl of ready to use master mix (Ampliqon, Denmark) contains (Tris-HCl pH 8.5, 1.5mM MgCl2, dNTPs and TaqDNA polymerase), 3 μl of genomic DNA samples and 10 μl distilled water.

PCR cycle parameters for PvLDH gene amplification were as follows: 5minutes initial denaturation at 95 °C followed by 30 cycles with 30 s at 95 °C, 30″ at 56 °C, 1′ at 72 °C and final extension at 72 °C for 5 min. All the PCR parameters were the same for PfLDH gene amplification except the annealing temperature was 58 °C. The PCR products of PvLDH and PfLDH were loaded on 1% agarose gel. The gel contained SimplySafe (EURx, Poland) for DNA staining. UV transilluminator was used to visualize the stained DNA. The fragment sizes of PCR products were determined using 1kb DNA ladder marker (Solis BioDyne, Estonia).

DNA sequencing

Twenty-two sequences including 15 P. vivax and 7 P. falciparum were analyzed to investigate polymorphism in PvLDH and PfLDH genes respectively. These genes were sequenced by applied biosystems 3730/3730xL DNA analyzers, (Bioneer, Seoul, Korea) using Sanger method. Nucleotide sequences of PvLDH and PfLDH were aligned and compared using Clustal W2 software (EMBL-EBI, http://www.ebi.ac.uk/Tools/msa/clustalw2/). PvLDH gene sequences were compared with GenBank sequences of P. vivax Belem (DQ060151.1) and P. vivax SaI-1 (XM_001615570.1). On the other hand, PfLDH gene sequences were compared with P. falciparum 3D7 (XM_001349953.1) and P. falciparum Mzr-1 (JN547219.1). Moreover, amino acid sequences related to each samples of P. vivax and P. faliparum were derived using ExPASy translate tool (http://web.expasy.org/tran-slate/). PvLDH amino acid sequences were compared with P. vivax SaI-1 (XM_001615570.1) and P. vivax Belem (DQ060151.1) whereas PfLDH amino acid sequences were compared with P. falciparum 3D7 (XM_001349953.1) and P. falciparum Mzr-1 (JN547219.1) strains registered in GenBank. Finally phylogenic tree was prepared to illustrate the distance among sequences of isolates using average distance (AD) method in Clustal W2 Jalview software (http://www.eb-i.ac.uk/). Thelactate dehydrogenase gene from Iranian Plasmodium strains were submitted with the accession numbers of (KM226649-KM226654 and KM226656-KM226664) for P. vivax, and (KM226665-KM226671) for P. falciparum in GenBank (BLAST).

Results

A 955 bp band was observed in gel electrophoresis of PCR products of PfLDH and PvLDH amplified genes (Fig. 1 and Fig. 2).

Fig. 1:

Gel electrophoresis of PvLDH gene PCR products. NC: Negative Control, Lane 1–3 PCR product samples, SM Size Marker (1kb)

Fig. 2:

Gel electrophoresis of PCR product samples of PfLDH gene. SM: Size Marker (1kb), NC: Negative control, Lane 1–4 PfLDH PCR products. Lane 3 did not show amplified gene

PvLDH genetic variation in Iranian isolates of P. vivax

The amplified PvLDH gene was yielded approximately 955 base pairs, coding for 316 amino acids. Fifteen of the amplified genes were sequenced to analyze the genetic variation of PvLDH gene using Clustal W2 software. After comparing the sequences with the chromatogram with P. vivax Sal-1 reference sequence, two Single nucleotide substitution were detected at 666, 899 positions from G to C and C to T respectively (Fig. 3).

Fig. 3:

Single-nucleotide substitution of pLDH gene among 15 P. vivax Iranian isolates, P. vivax Sal-1 (XM_001615570.1) and P. vivax Belem (DQ060151.1)

The nucleotide homology among Iranian isolates of P. vivax was 99.8–100%. Thirteen of the 15 isolates displayed 100% nucleotide sequence homology with P. vivax SaI-1 (XM_001615570.1) and P. vivax Belem (DQ060151.1) ( Table 1 & Fig. 4).

Table 1:

Comparing PvLDH nucleotide sequences of 15 Iranian isolates of P. vivax, P. vivax Sal-1 (XM_001615570.1) and Belem (DQ06015)

No.	Acc. No.	Plasmodium spp.	Isolates code	Nucleotide length (bp)	Homology rate (100%)
					Pv Sal-1 (XM_001615570.1)	Pv Belem (DQ060151.1)
1	KM226649	P. vivax	IB098	939	100	100
2	KM226650	P. vivax	IB099	939	100	100
3	KM226651	P. vivax	IB101	939	100	100
4	KM226652	P. vivax	IB105	939	100	100
5	KM226653	P. vivax	IB112	939	99.89	99.89
6	KM226654	P. vivax	IB115	939	100	100
7	KM226656	P. vivax	IB006	939	100	100
8	KM226657	P. vivax	IB063	939	100	100
9	KM226658	P. vivax	IB064	939	100	100
10	KM226659	P. vivax	IB067	939	100	100
11	KM226660	P. vivax	IB078	939	99.89	99.89
12	KM226661	P. vivax	IB081	939	100	100
13	KM226663	P. vivax	IB089	939	100	100
14	KM226664	P. vivax	IB090	939	100	100
15	KM226662	P. vivax	IB086	939	100	100

Fig. 4:

Amino acid alignment of PvLDH among Iranian P. vivax isolates (KM226649-KM226654) and (KM226656-KM226664), P. vivax Sal-1 and Belem (DQ060151.1)

The nucleotide substitution at 899 positions from C to T was brought an amino acid change from (T, neutral polar amino acid to me, non-polar amino acid) whereas the nucleotide substitution at 666 positions from G to C did not result any change in amino acid (Fig. 5). Fourteen Iranian isolates had 100% amino acid sequences with P. vivaxSaI-1andP. vivax Belem (Table 2).

Fig. 5:

Amino acid sequence difference in pLDH genes from Iranian P. vivax isolates (KM226649-KM226654) and (KM226656-KM226664), P. vivax Sal-1 (XM_001615570.10 and P. vivax Belem (DQ060151.1) using average distance (AD) tree from Clustal W2 Jalview software. Plasmodium berghei (AY437808.1) was used as an out group

Table 2:

Comparing amino acid sequences of PvLDH from Iranian isolates of P. vivax, P. vivax Sal-1(XM_001615570.1) and Belem (DQ060151)

No	Acc. No.	Plasmodium spp.	Isolates code	Nucleotide length (bp)	Homology rate (100%)
					Pv Sal-1 (XM_001615570.1)	Pv Belem (DQ060151.1)
1	KM226649	P. vivax	IB098	939	100	100
2	KM226650	P. vivax	IB099	939	100	100
3	KM226651	P. vivax	IB101	939	100	100
4	KM226652	P. vivax	IB105	939	100	100
5	KM226653	P. vivax	IB112	939	100	100
6	KM226654	P. vivax	IB115	939	100	100
7	KM226656	P. vivax	IB006	939	100	100
8	KM226657	P. vivax	IB063	939	100	100
9	KM226658	P. vivax	IB064	939	100	100
10	KM226659	P. vivax	IB067	939	100	100
11	KM226660	P. vivax	IB078	939	99.68	99.68
12	KM226661	P. vivax	IB081	939	100	100
13	KM226663	P. vivax	IB089	939	100	100
14	KM226664	P. vivax	IB090	939	100	100
15	KM226662	P. vivax	IB086	939	100	100

PfLDH genetic variation in Iranian isolates of P. falciparum

DNA was extracted from 10 P. falciparum confirmed whole blood samples and PfLDH gene was amplified using specific primers. Seven of the 10 amplified genes were sequenced and analyzed. DNA sequences of PfLDH gene displayed three nucleotide substitutions at 36, 814 and 891positions from A to G, G to A and G to A respectively (Fig. 6).

Fig. 6:

Nucleotide substitutions of pLDH gene among Iranian isolates of P. falciparum, P. falciparum 3D7 (XM_001349953.1) and P. falciparum isolates Mzr-1(JN547219.1)

The homology among PfLDH nucleotide sequences from Iranian isolates of P. falciparum were 99.67–100%. Five of the 7 isolates had 100% nucleotide homology with P. falciparum 3D7 (XM_001349953.1) and P. falciparum Mzr-1 (JN547219.1) strains submitted in GenBank (Table 3).

Table 3:

Comparing PfLDH nucleotide sequences of seven P. falciparum isolates with P. falciparum (XM_001349953.1) and P. vivax (JN547219.1)

No	Acc. No.	Plasmodium spp.	Isolates code	Nucleotide length (bp)	Homology rate (100%)
					Pf 3D7(XM_001 349953.1)	Pf Mzr-1(JN547219.1)
1	KM226665	P. falciparum	IF097	921	100	100
2	KM226666	P. falciparum	IF095	921	100	100
3	KM226667	P. falciparum	IF102	921	99.78	99.78
4	KM226668	P. falciparum	IF104	921	100	100
5	KM226669	P. falciparum	IF106	921	100	100
6	KM226670	P. falciparum	IF108	921	100	100
7	KM226671	P. falciparum	IF077	921	99.89	99.89

Acc. No.: Accession number, IF: Iran-Baluchistan falciparum

Only one of the nucleotide changes at 814 positions from G to A was brought an amino acid change from aspartic acid (D, acidic polar amino acid to N, neutral polar amino acid) (Fig. 7).

Fig. 7:

Amino acid sequence alignment of PfLDH among P. falciparum isolates, P. falciparum 3D7 (XM_001349953.1) and P. falciparum Mzr-1 (JN547219.1)

The rest six isolates showed 100% amino acid homology withPfMzr-1 and Pf3D7 strains from GenBank (Table 4).

Table 4:

Comparing PfLDH amino acid sequences of Iranian P. falciparum isolates, Pf3D7 (XM_001349953.1) and PfMzr-1(JN547219.1)

No	Acc. No.	Plasmodium spp.	Isolates code	Nucleotide length (bp)	Homology rate (100%)
					Pf 3D7(XM_001349 953.1)	Pf Mzr-1(JN547219.1)
1	KM226665	P. falciparum	IF097	921	100	100
2	KM226666	P. falciparum	IF095	921	100	100
3	KM226667	P. falciparum	IF102	921	100	100
4	KM226668	P. falciparum	IF104	921	100	100
5	KM226669	P. falciparum	IF106	921	100	100
6	KM226670	P. falciparum	IF108	921	100	100
7	KM226671	P. falciparum	IF077	921	99.69	99.69

Acc. No.: Accession number, IF: Iran-Baluchistan falciparum

PvLDH and Pf LDH homology from Iranian isolates of P. falciparum and P. vivax

The nucleotide homology between PvLDH and PfLDH in Iranian isolates of P. vivax and P. falciparum was 75.8–76%. All P. vivax LDH nucleotide sequences had 75.79% homology with six of P. falciparum isolates. The amino acids sequence homology between PvLDH and PfLDH Iranian isolates were 90.4% exception of one isolate which had 90.76% homology. Generally, the amino acids sequence homology between PvLDH and PfLDH Iranian isolates were more than 90%. PvLDH from Iranian isolates were also displayed 90.4 % homology with 3D7 and Mrz-1 P. falciparum isolates from gene bank.

Discussion

pLDH antigen is assumed to be a specific marker for the presence of viable Plasmodium in blood, and is used for screening in malaria-endemic countries (17). Inhibition of the malarial LDH enzyme prevents the producing ATP and causes death of the Plasmodium parasites,(13) so it becomes an attractive drug target candidate (16). The gene has the least diversity among Plasmodium spp. Therefore, the protein obtained from this gene can be used in any diagnostic test (18). Diversity in the pLDH gene may influence specificity and sensitivity of RDTs in any malaria endemic area. Investigation of polymorphism in P. vivax and P. falciparum lactate dehydrogenase gene can lead to produce more specific and sensitive RDTs kit.

The nucleotide homology among 15 PvLDH sequences of P. vivax was 100% with the exception of two isolates displayed 99.9% homology (Table 1, 2 & Fig. 5). In China, 100% PvLDH nucleotide sequence homology was reported among Chinese P. vivax, Sal-1 and Belem (19). However, our finding displayed two nucleotide substitutions. Another study done in China reported 99.89% nucleotide identity of Chinese isolates with Belem strain (20). This point out Iranian PvLDH nucleotide sequences had more homology with Belem strain than Chinese isolates.

Talman et al. reported PvLDH genes from Chinese P. vivax Anhui isolates had more than 99% sequence homology compared with strains in Gene bank (21). This outcome strongly agreed with findings from our study, which also showed more than 99% homology with all compared P. vivax strains registered in GenBank.

In the present study, PvLDH gene sequences showed two nucleotide substitutions with one resulted an amino acid change from T, neutral polar amino acid to I, non-polar amino acids. This substitution might not influence the sensitivity of PvLDH based RDTs. Antigen variability is unlikely to explain variability in implementation of RDTs detecting pLDH in P. falciparum, P. vivax cases (22). In contrast to our finding, Shin et al. in Korea reported one SNP which did not bring any change in amino acid (23) Jianget al. in China also reported a single nucleotide difference at the position 666 between PvLDH gene and P. vivax Belem (DQ060151)(24). The position of a nucleotide change in Jianget al. report was the same with one of the nucleotide substitutions detected in our study. PvLDH genes from Iranian isolates of P. vivax were displayed more nucleotide changes than Korean and Chinese isolates.

In earlier Chinese studies from Jianghuai region and Anhui isolates of P. vivax, there were no nucleotide changes among isolates (21, 25). Compared to these reports the nucleotide changes among PvLDH from Iranian isolates of P. vivax was higher than both Jianghuai region and Anhui isolates of P. vivax.

Talman et al. reported four different type of DNA sequence of P. vivax from 10 isolates; the mutations were synonymous (22). In our study, less number of nucleotide changes was seen and the mutations were not synonymous. Fourteen isolates had the same amino acid sequences with P. vivax SaI-1 (XM_001615570.1) and P. vivax Belem (DQ060151.1). This finding was agreed with a study conducted in China, which reported 100% PvLDH gene homology among Chinese isolates, P. vivax Sal-I and Belem (19). Studies done in Korea and China from Korean and Hainan isolates respectively, also reported 100% amino acid homology with P. vivax Belem (DQ060151.1) (20, 23). This indicated RDTs produced from Korean and Chinese isolates can be used in Iran.

On the other hand, PfLDH homology among Iranian strains of P. falciparum was 100% with the exception of two isolates. In contrast to our finding, Talman et al. reported no variability among all sequences P. falciparum (n = 49) in worldwide isolates of Plasmodium spp (22). This indicated PfLDH gene from Iranian isolates of P. falciparum had more nucleotide variation. Five of the seven isolates had 100% nucleotide identity with P. falciparum 3D7 (XM_001349953.1) and P. falciparum Mzr-1 (JN547219.1) strains registered in GenBank. Iranian PfLDH genes and reference sequence (Pf3D7) had high homology about 99.9%–100% (Table 1, 2 & Fig. 8).

Fig. 8:

Amino acid sequence difference in pLDH genes from P. falciparum Iranian isolates, P. falciparum 3D7 (XM_001349953.1) and P. falciparum Mzr-1 (JN47219.1) using average distance (AD) tree. Plasmodium berghei (AY437808.1) was used as an out-group

This indicated PfLDH gene is relatively conserved and can be a good target for antimalarial drug and producing RDT. In our study, six of the 7 isolates had the same amino acid sequence. These amino acid sequences also had 100% homology with P. falciparum Mzr-1 and P. falciparum 3D7 strains from Gen-Bank. Our finding was supported by the study conducted in Indonesia, which reported 100% amino acid sequences between Indonesian PfLDH and Pf 3D7 reference sequence (26).

In this study, two isolates displayed three nucleotide substitutions at 36, 814 and 891 positions. However, only the substitution at 891 positions from G to A was brought an amino acid change from aspartic acid to asparagine (D, acidic polar amino acid to N, neutral polar amino acid). In Madagascar, two SNPs at 73 and 814 positions among the 137 DNA sequences of P. falciparum isolates were displayed. Both single nucleotide polymorphisms (SNPs) in Madagascar study brought amino acid changes. The nucleotide change in 10 isolates at 814 position resulted in an amino acid change (D, acidic polar amino acid to N, neutral polar amino acid). In addition, another amino acid change (at codon 25: Q, neutral polar amino acid to A, basic polar amino acid) was seen due to the SNP at 73 position (18). The position of nucleotide change (814bp) and the resulted amino acid change (aspartic acid (D) to asparagine (N) in one of the isolates in our study was the same with the Madagascar study. The nucleotide change at 814 positions in our study might be a single nucleotide polymorphism given the Madagascar study SNP report at the same position. Iranian PfLDH demonstrated less number of amino acid changes in comparison with the report that released from Madagascar study. The nucleotide sequences homology between Iranian isolates of PvLDH and PfLDH were 75.79–76%. In China, Jiang et al. was reported 75.1% homology between PvLDH and PfLDH nucleotide sequences (24). Akbulut et al. also reported 74.8% homology between PvLDH and PfLDH (27). Compared to Jiang et al. and Akubulut et al. report, the homology of Iranian PvLDH and PfLDH was high. In our study, the amino acid sequences homology among Iranian isolates of PvLDH and PfLDH was 90.4% with the exception of one isolate. Shin et al. and Turgut-Balik et al. reported 89.5% and 90.2% amino acid sequence homology between PvLDH and PfLDH respectively (23, 28). This indicated the amino acid homology between PvLDH and PfLDH genes from Iranian isolates of P. vivax and P. falciparum were higher than previously reports. Generally, in our study the amino acid homology between PvLDH and PfLDH was more than 90%. This was supported by Turgut-Balik et al. report (28).

Conclusion

pLDH gene from Iranian P. falciparum and P.vivax isolates displayed 98.8–100% homology with 1–3 nucleotide substitutions. This relatively stability indicated PvLDH and PfLDH genes can be a good antimalaria target and used for producing RDT kits. The amino acid sequence homology of PvLDH and PfLDH was more than 90%. This indicated some techniques like drug discovery, vaccine development and other activities, which were applied on P. falciparum, could also be tried for P. vivax. The homology among pLDH of P. vivax and P. falciparum should be further investigated with large enough sample size. In general, before using pLDH for producing RDT kits the genetic variation of this gene should be investigated since its polymorphism varies with geographical locations.