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Genetic diversity of msp3α and msp1_b5 markers of Plasmodium vivax in French Guiana

Background Reliable molecular typing tools are required for a better understanding of the molecular epidemiology of Plasmodium vivax. The genes msp3a and msp1_block5 are highly polymorphic and have been used as markers in many P. vivax population studies. These markers were used to assess the genetic diversity of P. vivax strains from French Guiana (South America) and to develop a molecular typing protocol. Methods A total of 120 blood samples from 109 patients (including 10 patients suffered from more than one malaria episode, samples were collected during each episode) with P. vivax infection were genotyped. All samples were analysed by msp3a PCR-RFLP and msp1_b5 gene sequencing was performed on 57 samples. Genotyping protocol applied to distinguish between new infection or relapse from heterologus hypnozoites and treatment failure or relapse from homologus hypnozoites was based on analysing first msp3a by PCR-RFLP and secondly, only if the genotypes of the two samples are identical, on sequencing the msp1_b5 gene. Results msp3a alleles of three sizes were amplified by PCR: types A, B and C. Eleven different genotypes were identified among the 109 samples analysed by msp3a PCR-RFLP. In 13.8% of cases, a mixed genotype infection was observed. The sequence of msp1_b5 gene revealed 22 unique genotypes and 12.3% of cases with mixed infection. In the 57 samples analysed by both methods, 45 genotypes were found and 21% were mixed. Among ten patients with two or three malaria episodes, the protocol allowed to identify five new infections or relapses from heterologous hypnozoites and six treatment failures of relapses from homologous hypnozoites. Conclusion The study showed a high diversity of msp3a and msp1_b5 genetic markers among P. vivax strains in French Guiana with a low polyclonal infection rate. These results indicated that the P. vivax genotyping protocol presented has a good discrimination power and can be used in clinical drug trials or epidemiological studies.


Background
Malaria remains one of the most serious vector diseases in the world, and there are between 300 to 500 million cases of malaria annually. Although considered less dangerous than Plasmodium falciparum, Plasmodium vivax infections occasionally cause fatal cases [1][2][3][4] and have a large economic impact in endemic countries. There are estimated to be 70-80 million cases of P. vivax infection annually [5].
French Guiana, an overseas French territory in equatorial South America, suffers endemic malaria with areas of moderate and high transmission. The epidemiological profiles in the two main endemic foci differ. Plasmodium falciparum is largely predominant along the Maroni River, western border with Surinam, particularly in "Bush Negro territory". Plasmodium vivax is more common in the Oyapock focus, eastern border with Brazil and in the eastern slightly inland areas with a substantial increase since mild 2001 [6]. Plasmodium malariae is much rarer. The inland regions of French Guiana, located between the two Border Rivers, are uninhabited with the exception of two villages. In coastal regions where three quarters of the population (205,000 inhabitants according the 2008 official censuses) live, occasional bouts of malaria, mainly imported malaria, are recorded.
Plasmodium vivax is characterized by relapses at various time intervals associated with the presence of hypnozoites. Investigations of relapses of P. vivax infection report activation of heterologous hypnozoites in East Timor [7], Thailand and India [8]. There are genetic differences between the P. vivax populations originating from these two continents [9,10]. So far, there are few available data on hypnozoite activation during P. vivax relapse in patients in South America. Therefore, a better genotypic characterization of P. vivax at a regional scale is necessary [11]. To develop reliable epidemiological tools, appropriate genetic markers and practical molecular techniques are needed. Althought microsatellites are the most highly discriminating markers, their development is laborious and their analysis costs expensive. Merozoite Surface Proteins (MSP) have been extensively studied. These proteins of the erythrocytic stage of the Plasmodium life cycle are potential targets for vaccine and can also be used for genetic typing [12]. Pvmsp3a and Pvmsp1 are single copy genes that are highly polymorphic and currently used for P. vivax genotyping [13]. The MSP3a protein has a molecular weight of between 148 and 150 KD, and the highly polymorphic region of the gene encodes an alanine-rich central domain. Nucleotide polymorphism of msp3a has been studied by PCR-RFLP in different geographical regions (Iran and Papua New Guinea [14][15][16][17]) and has also been analysed by gene sequencing in Venezuela and Thailand [18,19]. Pvmsp1, another well-studied gene [9,12,[20][21][22][23][24][25][26], encodes a protein of approximately 1,720 amino acids, and sequence comparisons indicate that the gene is found as two major sequence types, known as Belem and Salvador [27]. Interallelic recombination of these two sequences has been also described [22]. The Pvmsp1 gene is long, so many genetic studies have focused on block 5, a polymorphic region suitable for molecular typing.
The objectives of the present study were firstly to analyse the genetic polymorphism of Pvmsp3a and Pvmsp1_b5 markers in French Guiana; and secondly to propose a protocol for distinguishing between new infection or relapse from heterologus hypnozoites and treatment failure or relapse from homologus hypnozoites. The protocol was tested with blood samples from patients with multiple malaria episodes.

Samples
A total of 120 venous blood samples were collected in EDTA-tubes during 2005 and 2006 from 109 symptomatic malaria patients. Samples were collected as part of routine diagnosis conducted for 52 patients consulting the Health Centers at Saint Georges, Camopi and Trois Sauts living in the malaria Oyapock focus (semi-immune population) and for 57 patients consulting the Emergency Service of Cayenne (mainly non immune patients). For ten patients (R001 to R010) included in the 57, two (and for R006 three) blood samples were studied.
msp3a RFLP was performed on all samples, the b5 region of msp1 was sequenced from the patients seen in Cayenne Hospital. All samples had been sent to the malaria reference laboratory at Institut Pasteur de la Guyane by health centres (of St Georges, Camopi and Trois Sauts) and by the Cayenne Hospital, for expertise and typing as recommended by the health authorities for malaria survey.
Plasmodium vivax treatment was mainly chloroquine alone, as the use of primaquine was not widely authorized in 2005 -2006, on administrative grounds.

DNA extraction
Plasmodium vivax genomic DNA was extracted from 100 μL of EDTA-treated whole blood with DNeasy Blood and Tissue kits (Qiagen, Hamburg, Germany) following manufacturer's instructions. DNA was eluted in 200 μL of Tris-EDTA buffer and kept at + 4°C until use or stored at -20°C.

Genotyping of P. vivax by msp3α PCR-RFLP
Msp3a polymorphism was studied using the PCR-RFLP method described by Bruce et al [5]. The msp3a gene was amplified by nested PCR in a final volume of 25 μL; 1 μL of DNA extract was used for the first PCR, and 1 μL of the first reaction product was used in the second PCR. Seven microliters of each second PCR product was digested individually with 5 units of HhaI enzyme (New England Biolabs Inc, UK) by incubation at 37°C for 3 hours in a final volume of 20 μL in buffer supplied with the enzyme. DNA fragments were visualized under UV illumination after electrophoresis on 1.8% agarose gel (W/V) containing 0.25 μg/mL of ethidium bromide. Polyclonal infection can be identified either by observing on electrophoresis more than one allele of the undigested PCR product (type A, B or C) or by comparing the sum of the sizes of digested fragments with the size of the uncut fragment [7].

Genotyping of P. vivax by msp1_b5 gene sequencing
The DNA fragment encompassing the ICB5-ICB6 region of the msp1 gene was amplified by PCR as described by Premawansa et al [15]. Briefly, the amplifications were performed in 50 μL reaction buffer containing the DNA template, 1.5 mM MgCl 2 , 2 μM of each primer ICB5 (5' CCT ACT ACT TGA TGG TC) and ICB6 (5' CCT TCT GGT ACA GCT CAA TG), 200 μM of dNTP and 1.5 unit of AmpliTaq Gold polymerase (Applied Biosystem, Foster City, CA, USA). The PCR products were visualised by 1% agarose gel electrophoresis in the presence 0.25 μg/mL of ethidium bromide. Positive amplifications were purified using Qiaquick Gel Extaction Kits (Qiagen, Hamburg, Germany) and analysed by direct double strand sequencing using BigDye Terminator Kit (Applied Biosystem, Foster City, CA) with ICB5 and ICB6 primers. Sequences reactions were performed by the society Millegen (Labege, France). The amino acid sequences were aligned with the corresponding Belem and Salvador reference sequences (GenBank accesion number: M60807 and AF435603 respectively) by using the Clustal W program [28].

Distinction between new infection or relapse from heterologous hypnozoites and treatment failure or relapse from homogous hypnozoites
Two samples corresponding to sequential of malaria from a patient are first analysed by PCR-RFLP: i) if the genotypes found are different, this identifies the second episode as a new infection or a relapse from heterologous hypnozoites, and there is no need for further analysis; ii) if the genotypes are identical, msp1_b5 sequence should be determined; iii) if samples are of different msp1_b5 type, this identifies the second episode as a new infection or a relapse from heterologous hypnozoites; iv) if same genotypes are found, treatment failure or relapse from homologous hypnozoites can be concluded with a risk of error proportional to the allele occurrence.
This protocol was assessed for 10 patients, two (or three for R006) samples were collected during successive malaria episodes at intervals of 18 days to six months.

Diversity of the Pvmsp3a gene
The Pvmsp3a gene was amplified from total parasite DNA obtained from 109 patients. PCR products of three lengths were found: 1.9 kb (type A) corresponding to the published sequence of the Belem strain, 1.5 kb (type B) and 1.1 kb (type C). HhaI digestion indicated that 15 (13.8%) of the 109 blood samples were polyclonal. The frequencies of the allelic groups in mono-infected samples (n = 94) were 85.1% for type A, 9.6% for B and 5.3% for C.
In cases of polyclonal infections, genotype identification was not possible. The PCR-RFLP patterns of all three genotypes included an approximately 1.0 kb band which was slightly polymorphic. Consistent with Mueller et al [7], this band was not used to distinguish patterns because the size differences were not easily resolved by agarose gel electrophoresis. HhaI digestion revealed nine different genotypes (A1 to A9) in allelic group A, and only one genotype in each group B and group C ( Table 1). Genotypes A1, A4 and A7 were the most numerous with apparition frequencies of 27.7, 12.8 and 13.8%, representing 54.3% of the monoclonal samples; the other six genotypes (A2, A3, A5, A6, A8 and A9) together made up 30.8% of the monoclonal infections.

Analysis of Pvmsp1_b5 gene sequence
The msp1_b5 gene was amplified from total parasite DNA ( Table 2). Amplified fragment lengths were between 630 and 720 base pairs. Seven (12.3%) of the 57 samples contained two different msp1_b5 sequences indicating polyclonal infection. A total of 22 different sequences were found, belonging to the three major sequence types: Note: As genotypes of polyclonal samples were not clearly identified, they were not be integrated in the frequency calculation.

Analysis of samples with the two genotyping methods
Analysis of all the data provided by both genotyping methods showed 45 different genotypes among 57 analysed samples, including 12 polyclonal infections (21%) ( Table 3). Twelve of the 57 samples were A1 type; msp1 sequencing of these 12 samples gave four of genotype H1A, two H2B, two mixed (H1A + H2B and SAL-1 C + Belem3), one of each H1D, SAL-1 A, SAL-1 H and SAL-1 I. Among the five A4 samples, msp3a genotyping found four different msp1 genotypes, two samples being H1A. The six A7 genotype samples were all different msp1 alleles.
The two genotyping methods did not identify the same polyclonal infections: there were twelve (21%) such cases, and msp3a genotyping identified nine (15.8%) and msp1 sequencing seven (12.3%); thus only four patients were identified as having polyclonal infections by both techniques.

Genotyping of patients with multiple bouts of malaria
For 10 patients, two (or three for R006) samples were collected during successive malaria episodes at intervals of 18 days to six months ( Table 4). The patients lived in the littoral zone and were contaminated during a stay along the Oyapock river. They have received a treatment with chloroquine, which has no effect on the exo-erytrocytic forms. An another stay in endemic zone, between malaria episodes, could not be excluded.
Msp3a genotypes of successive samples from patient R001 and R004 were different, indicating that these second episodes were due to a new infection or a relapse from heter-  infection or a relapse from heterologous hypnozoites (msp1 genotype found: SAL-1 B).

Discussion
Plasmodium vivax diversity has been less extensively studied than that of P. falciparum. A better characterization of P. vivax populations in South America was particularly lacking. A large number of samples must be analysed to describe the genetic structure of a population, so genetic markers that are quickly and easily detectable at a reasonable cost are required. The aims of this work were to study the polymorphism of msp3a and msp1_b5 markers and to develop a method for distinguishing between new P. vivax infections or relapses from heterologous hypnozoites and treatment failure or relapse from homologous hypnozoites. It is of crucial importance clinically when selecting drug treatment, and in particular chosing between drugs with or without effect on the exo-erythrocytic stages. msp3a and msp1 block 5 sequence analysis indicated a high degree of polymorphism, according to different continents and countries, making these genes informative for molecular epidemiological studies. Nevertheless, they also contain conserved regions allowing amplification of the nucleotide sequences of interest. The polymorphism of msp3a can be evaluated rapidly at low cost by PCR-RFLP [14][15][16][17]. Msp1_b5 sequence reveals substantial diversity [9,12,[20][21][22][23][24][25][26][27]. The RFLP technique allows a better discrimination of polyclonal infection samples than sequencing [29], so this protocol associating RFLP and sequencing could be used in regions with high rates of polyclonal infections.
Work on relapses of P. vivax infections demonstrates activation of heterologous hypnozoites. The genotype of the acute case is not the same as that of the recurrent infection in about 71% of relapses observed in Australian soldiers returning from East Timor [7], 61% of Thai and Burmese patients and 55% of Indian patients [8].
In French Guiana, 11 different msp3a genotypes were identified among 109 samples and 13.8% had mixed-species infections. Polyclonal infections were thus less frequent than described by Bruce et al and Cui et al [13,14], who reported 23% in Papua New Guinea and 19.3% of in Thailand, respectively. Three previously described types were found: in 85.1 (type A), 9.6 (type B) and 5.3% (type C) of the monoclonal samples.  [18], and type C has been reported to be much more frequent in southern Iran [15] than in the studied population. Over half (54.3%) of the isolates carried one of only three alleles, A1, A4 and A7, with the other eight genotypes accounting for the remaining 45.7% of the monoclonal samples.
Polymorphism was also studied of the msp1_b5-6 sequence. For this protocol, genotyping msp1 is required in cases for which the msp3a genotype is identical in consecutive samples. Twenty-two different sequences in the 57 samples tested were observed. Four genotypes were strongly represented: H1A, H2B, SAL-1 E and SAL-1 H made up 51.5% of the alleles and 25% overall were H1A. The frequencies of the other 18 alleles were between 4.7 and 1.7%. A study in Iran of msp1_b5 sequences in 145 isolates reported 30 genotypes [26]. Seven (12.3%) mixed infections by msp1 sequencing, and like msp3a polymorphism analysis, this suggests that polyclonal infections are less frequent here than in Papua New Guinea with 38% [20]. The possible causes for this difference include malaria endemicity, population and vector density.
From these data a maximal msp1 allelic frequency of 5%, at which two samples from a same patient found with the same genotype can be considered to be a treatment failure or a relapse from homologous hypnozoites. In other words, relapse should be concluded with a reasonable risk of error up to 5%. If the genotype found is more represented, error risk in the conclusion of the analysis will be proportional to the genotype occurrence found.
The two methods combined revealed 45 genotypes among the 57 samples and 12 (21%) cases of polyclonal infection. Only four of these cases of polyclonal infection were identified as such by both genotyping methods and eight were identified only by one technique. Five patients were found by msp3a PCR-RFLP to have polyclonal infection, but msp1 sequencing suggested mono-infection. In cases of mixed infection with one genotype being very poorly represented in the biological sample, sequencing may not easily detect the minority genotype. In contrast, msp3a PCR involving nested amplification is more sensitive, and thus an additional allele should be more easily detected as it will give a 'parasite' band in addition to the known PCR band or known RFLP pattern. Thus, PCR-RFLP seems to be more sensitive than direct sequencing for detecting polyclonal infections [29]. Inversely, three patients were detected with a polyclonal infection with msp1 sequencing although msp3 PCR-RFLP detected only one genotype (two with genotypes A1 and one with A7). As the frequencies of msp3a genotypes A1 and A7 were high (27.7 and 13.8%, respectively), it is plausible that the two msp1 genotypes in these samples have the same msp3a genotype. The polymorphism of Pvmsp1_b5 and Pvmsp3a as described in many parts of the world, has been observed in French Guiana.
Samples from ten patients who had suffered two or three bouts of malaria (R001 to R010) were analysed. Five episodes were considered to be new infections or relapse from heterologous hypnozoites (patients R001, R002, R004, R005 and third episode for R006). Six of the second bouts were identified as being treatment failures or relapses from homologous hypnozoites. This was concluded for three patients with less than 5% risk of error and with more than 5% for also three others. The time interval between episodes for these six patients was 27 to 50 days. Molecular typing methods cannot distinguish between the both outcomes and treatment failure can occurs one month after the first episode. Therefore, based on these data, the question remains open and it is of crucial importance in clinical trials in endemic areas to know the cause of the latter episode. Moreover, to be sure that in sequential patient sample genotypes are identical, it should be verified using more efficient markers, such as microsatellites.

Conclusion
A high diversity of msp3a and msp1_b5 genetic markers was observed among P. vivax strains in French Guiana and revealed a low polyclonal infection rate. The described protocol associates two molecular techniques for P. vivax genotyping: msp3a PCR-RFLP, which is a low cost method based on a highly polymorphic gene, and msp1_b5 sequencing, a highly discriminatory genetic tool, which provides complementary genotyping data, if required. The methodology can be easily used as a moderate-or highthroughput analysis protocol for clinical trials or epidemiological studies.