- Open Access
Genetic analysis of the merozoite surface protein-1 block 2 allelic types in Plasmodium falciparum clinical isolates from Lao PDR
- Naly Khaminsou†1,
- Onanong Kritpetcharat†2,
- Jureerut Daduang†3,
- Lertchai Charerntanyarak4 and
- Panutas Kritpetcharat5Email author
© Khaminsou et al; licensee BioMed Central Ltd. 2011
- Received: 16 August 2011
- Accepted: 17 December 2011
- Published: 17 December 2011
MSP-1 is one of the potential malarial vaccine candidate antigens. However, extensive genetic polymorphism of this antigen in the field isolates of Plasmodium falciparum represents a major hindrance for the development of an effective vaccine. Therefore, this study aimed to establish the prevalence and genetic polymorphisms of K1, MAD20 and RO33 allelic types of msp-1 block 2 among P. falciparum clinical isolates from Lao PDR.
Plasmodium falciparum isolates were collected from 230 P. falciparum-infected blood samples from three regions of Lao PDR. K1, MAD20 and RO33 were detected by nested PCR; SSCP was used for polymorphism screening. The nested PCR products of each K1, MAD20 and RO33 allelic types that had different banding patterns by SSCP, were sequenced.
The overall prevalence of K1, MAD20 and RO33 allelic types in P. falciparum isolates from Lao PDR were 66.95%, 46.52% and 31.30%, respectively, of samples under study. Single infections with K1, MAD20 and RO33 allelic types were 27.83%, 11.74% and 5.22%, respectively; the remainders were multiple clonal infections. Neither parasite density nor age was related to MOI. Sequence analysis revealed that there were 11 different types of K1, eight different types of MAD20, and 7 different types of RO33. Most of them were regional specific, except type 1 of each allelic type was common found in 3 regions under study.
Genetic polymorphism with diverse allele types was identified in msp-1 block 2 among P. falciparum clinical isolates in Lao PDR. A rather high level of multiple clonal infections was also observed but the multiplicity of infection was rather low as not exceed 2.0. This basic data are useful for treatment and malaria control program in Lao PDR.
- Parasite Density
- Allelic Type
- Falciparum Isolate
- Vaccine Candidate Antigen
Malaria remains one of the most important health-threatening parasitic diseases in tropical and subtropical areas, such as Lao PDR and other countries in Southeast Asia. In Lao PDR, many highly endemic areas exist, especially in the rural areas of Xekong, Attapeu, Savannakhet, Saravane, and Champasack provinces [1–3]. Plasmodium falciparum is responsible for most of the mortality, while Plasmodium vivax causes considerable morbidity [1, 3, 4]. Despite the enormous efforts that have been directed toward malaria control and prevention, multiple factors-including insecticide resistance in anopheline vectors, the lack of effective vaccines, and the emergence and rapid spread of drug-resistant strains-are the major problems for controlling and prevention of malaria. Therefore, the development of an effective malaria vaccine is urgently needed. However, extensive the genetic diversity in natural malaria parasite populations is a major obstacle for the development of an effective vaccine against these parasites, because antigenic diversity limits the efficiency of acquired protective immunity to malaria [5–7]. Many malarial proteins have been proposed for use as vaccine candidate antigens, but merozoite surface protein-1 (MSP-1) is the most used [8, 9]. However, extensive genetic polymorphisms of the MSP-1 gene have been identified in P. falciparum isolates worldwide; this has caused extensive antigenic polymorphism [10, 11]. It is important to investigate the diversity of msp-1 gene, in different geographic areas for the further development of effective malaria prevention and control.
MSP-1, with an approximate molecular size of 190 kDa, is a major surface protein of P. falciparum, and plays a role in erythrocyte invasion by the merozoite . The protein is a principal target of human immune responses [13–15] and is a vaccine candidate antigen for blood stages [12–16]. The msp-1 gene has 7 variable blocks that are separated either by semi-conserved or conserved regions. Block 2, a region near the N-terminal of this gene, is the most polymorphic part of the protein and appears to be under the strongest diversifying selection within natural populations . At present, four different allelic types of block 2 have been identified, including K1, MAD20, RO33 and MR [17, 18].
The overall prevalence of malarial infection in Lao PDR recoded in 2008 by the Central of Malaria, Parasitology and Entomology of Lao PDR was 15% [Unpublished data]. At present, there is no data about the genetic diversity of P. falciparum in Lao PDR. For this reason, it is important to investigate the diversity of msp-1 gene, in different geographic areas for the further development of effective malaria prevention and control.
This study aimed to establish the prevalence and genetic polymorphisms of K1, MAD20 and RO33 allelic types of the msp-1 gene among P. falciparum field isolates from Lao PDR.
Blood samples and genomic DNA extraction
Demographics of the studied population
Oudomxay No. (%)
Savannakhet No. (%)
Xekong No. (%)
Total No. (%)
Nested PCR for Plasmodium falciparum
All extracted DNA samples were confirmed for Plasmodium infection by using nested PCR, as described by Snounou et al. . Genus-specific primers and species-specific primers of P. falciparum, P. vivax, Plasmodium malariae and Plasmodium ovale were used in this study. Blood samples infected by Plasmodium species other than P. falciparum, or by mixed species, were excluded.
Nested PCR for msp-1 block 2, SSCP for polymorphism screening and DNA sequencing
Sequences of oligonucleotide primers used to amplify msp-1 block 2 (K1, MAD20 and RO33) allelic types of Plasmodium falciparum 
The first round of PCR was done with the external 5' and 3' primers, which amplified msp-1 block 2 plus some of the flanking regions. The first round of amplification was carried out in 25 μl of reaction mix containing: 1× PCR, 4.0 mM MgCl2, 0.1 mg/ml gelatin, 0.05% Triton X-100, 200 μM dNTP mix, 0.8 U Taq polymerase, 75 nM of each conserved primer, distilled water up to 20 μl, and 5 μl of DNA template. Amplification was performed using the following conditions: 94°C for 5 min, 35 cycles at 94°C for 30 s, 58°C for 1 min, and 72°C for 1 min, followed by extension at 72°C for 10 min.
The second round of PCR was performed in three separate tubes each containing a single pair of K1, MAD20 and RO33 allele-specific primers. The 25 μl reaction mix contained 1× PCR, 4.0 mM MgCl2, 0.1 mg/ml gelatin, 0.05% Triton X-100, 200 μM dNTP mix, 0.8 U Taq polymerase, 75 nM of each allele-specific primer pair (K1a/K1b, MAD20a/MAD20b or RO33a/RO33b), distilled water up to 20 μl, and 5 μl of the first-round PCR product. Amplification cycles for second round PCR were initial denaturation for 5 min at 94°C, followed by 35 cycles of 30 s denaturation at 94°C, 1 min annealing at 58°C, and extension at 72°C for 1 min. Final extension was carried out at 72°C for 10 min. PCR products were visualized by UV transillumination at 302 nm on gel documentation system after electrophoresis on 2% agarose gel (Promega/Boe-hringer) using 0.5 × TBE buffer at 100 volts.
Each DNA product was screened for polymorphic banding using SSCP, as described in Huby-Chilton et al. , in duplicate: one stained with silver nitrate and the other with ethidium bromide. The K1, MAD20 and RO33 products from nested PCR that showed normal and polymorphic bands were sent for DNA sequencing by using each forward and reverse primer pair.
Statistical and DNA sequence analysis
A two-proportions test was used to compare the prevalence of msp-1 block 2 allelic types between age groups, between genders and between parasitaemic groups. To understand the identity of msp-1 block 2 allelic types of Lao PDR with respect to isolates of other regions worldwide, sequence data available in the public domain were downloaded for the following msp-1 allelic types: 3D7 (K1-XM 001352134); Thailand (K1-AB276018, K1-AB276008, MAD20-AB276013, MAD20-AB276006, RO33-AB276015, RO33-M77737); Vietnam (K1-AF509651, MAD20-AF509699, MAD20-AF509696, MAD20-AF509694); Cambodia (K1-HM153247, MAD20-HM153249); Indonesia (K1-AF191061, RO33-AF191064); India (K1-DQ485421, K1-DQ485424, RO33-JF300128); Myanmar (K1-GQ861445, K1-EU445566, MAD20-EU445560, MAD20-EU445555); Ghana (RO33-AB276005); Malawi (K1-HM153200, MAD20-HM153237, RO33-HM153239, RO33-HM153223); Gambia (MAD20-AB276004); Peru (K1-FJ612037, K1-FJ612017, RO33-FJ612064); Tanzania (MAD20-AF061147, RO33-AF061150); Brazil (MAD20-AF509667, RO33-AB276002); Iran (RO33-AY138508); Sudan (RO33-AB300615); and western Africa (RO33-M55001). DNA sequence data for each allelic family was aligned together with other allelic sequences worldwide by using the ClustalW program through BioEdit 5.0.7 software; phylogenetic trees were created using MEGA 5 .
The distribution of msp-1 block 2 allelic types in P. falciparum field isolates from Lao PDR was determined, based on microscopic diagnosis, from a total of 230 blood samples randomly collected from P. falciparum-infected patients who attended regional malarial clinics in three geographic areas of Lao PDR from July 2008 to May 2009: in Oudomxay province (representing the northern region), Savannakhet province (central), and Xekong province (southern). The patients consisted of 116 (50.43%) males and 114 (49.57%) females: 76 (33.04%) children younger than 5 years of age; 94 (40.87%) patients whose ages ranged 5-19 years; 39 (16.96%) patients whose ages ranged 20-39 years; 19 (8.26%) patients whose ages ranged 40-59 years; and 2 (0.87%) patients whose ages ≥ 60 years. These blood samples were confirmed for P. falciparum infection.
Prevalence of msp-1 block 2 allelic types in Lao PDR
msp-1 block 2 allelic type
Oudomxay No. (%)
Savannakhet No. (%)
Xekong No. (%)
Total No. (%)
K1 + MAD20
K1 + RO33
MAD20 + RO33
K1 + MAD20 + RO33
Distribution of K1, MAD20 and RO33 allelic types among age groups
msp-1 block 2 allelic type
Age group (years)
< 5 No. (%)
5-19 No. (%)
20-39 No. (%)
40-59 No. (%)
≥ 60 No. (%)
Total No. (%)
K1 + MAD20
K1 + RO33
MAD20 + RO33
K1 + MAD20 + RO33
Distribution of msp-1 block 2 allelic types among parasitaemic groups from 3 regions of Lao PDR
msp-1 block 2 allelic type
Number of cases (%)
K1 + MAD20
K1 + RO33
MAD20 + RO33
K1 + MAD20 + RO33
The polymorphic alleles of K1, MAD20, and RO33 results by SSCP screening of P. falciparum field isolates from Oudomxay, Savannakhet, and Xekong
msp-1 block 2 allelic type
% (polymorphic alleles/total)
Total polymorphic alleles
Human malaria is an infectious disease transmitted by the bite of mosquitoes infected with any of five Plasmodium species: P. falciparum, P. vivax, P. malariae, P. ovale and Plasmodium knowlesi. Among these species, P. falciparum can cause various clinical problems including heart, lung, kidney and brain damage, and possibly death. P. falciparum remains an important public health concern because of its increasing resistance to common anti-malarial drugs, resulting in a high mortality rate. In malaria-endemic areas, infections by multiple parasite clones are frequent, and clone fluctuation may be a logical strategy for the appearance of strain-specific responses [25–28].
This study investigated the prevalence and polymorphism of P. falciparum subpopulations in three regions of Lao PDR by nested PCR. This nested PCR method cannot be used to distinguish alleles nearly identical or identical in size, but differing in sequences . Polymorphic banding by size of all parasite isolates in this study was not observed by agarose gel electrophoresis of the nested PCR products because all isolates were the same or very nearly the same size. To distinguish polymorphism in allele-specific families in this study, all DNA products from each allele-specific family detected by nested PCR were screened by SSCP. The different band patterns from SSCP were selected, and these nested PCR products were sequenced. This study found that sequences that had only one nucleotide base change could produce a different banding pattern on SSCP, as seen in Xekong isolate K1 type 10 and Savannakhet isolate MAD20 type 4. Moreover, nested PCR could not detect P. falciparum isolates, which have polymorphic allele sequences because of insertion or deletion of a few nucleotide bases. The prevalence of polymorphic strains found in each msp-1 block 2 allelic type in this study may be underestimated because of variations in sensitivity which occurred throughout the experiment due to weak bands, as well as in the estimation of band length, as suggested by Aubouy et al. .
Nevertheless, the P. falciparum isolate K1 allelic type was found to be the most prevalent in Lao PDR, as is the case in other regions worldwide: K1, MAD20 and RO33 allelic types comprised 66.95%, 46.52% and 31.30%, respectively, of samples in the present study. When observing the three allelic types in the three regions of Lao PDR separately, this study found that the MAD20 allelic type was more prevalent than the others in Oudomxay, the least malaria-endemic area of Lao PDR. However, the number of samples collected from Oudomxay was low due to the sampling formula, which depended on the prevalence of malaria infection in this region. The distribution of K1, MAD20 and RO33 allelic types in highly endemic areas such as Savannakhet and Xekong provinces had the same patterns and the same overall distribution as the total three regions.
Monomorphic banding of K1 and MA20 allelic types in Lao PDR observed on agarose gel electrophoresis may be due to limited samples group with clinical symptoms in this study, or due to the fact that clonal fluctuations in each msp-1 block 2 allelic types of P. falciparum isolate in Lao PDR is quite low. Moreover, it may be due to low parasite density in the blood samples under study (majority of parasite density < 1,000 cells/μl). Limited clonal fluctuation of K1 allelic type was also seen in urban malaria infected patients (200 and 220 bp) in Burkina Faso  and in Darjeeling district, West Bengal state of India (140 bp) . Moreover, monomorphic band of MAD20 (220 bp) and RO33 (150/160 bp) were also found in some districts in eastern and northeastern India .
However, the total P. falciparum msp-1 block 2 genotypic isolates, multiple clonal infections and MOI in this study might be underestimated, because the new MR allele type, as described by Takala et al. , was not investigated. Takala et al. reported that the MR allele type in P. falciparum isolates in Asembo Bay, Kenya, was found in 29% of the samples. Happi et al.  found a proportion of K1 (79%), MAD20 (32%) and RO33 (38%) alleles in P. falciparum isolates from Nigeria. The frequency of MAD20 in the study by Happi et al. was lower than in this study and in studies from other regions. Aubouy et al.  studied the polymorphism in two MSP-1 and MSP-2 genes from Gabon. They found that MSP-1 alleles of K1, MAD20 and RO33 comprised 90.4%, 63.5% and 36.5% of the samples studied; they also found that P. falciparum polymorphism was extensive in southeast Gabon, and that most infections were composed of multiple clones. About 45.65% (105/230) of P. falciparum infections in Lao PDR in this study were multiple clonal infections of the three allele types studied. Kang et al.  studied genetic polymorphism of msp-1 and msp-2 in P. falciparum field isolates from Myanmar, and found that the K1 allele infection rate was 73.02% (46/63); but the majority (63.5%) occurred as multiple clonal infections with the MAD20 allele type. This study found that total multiple clonal infections of K1 and MAD20 (including K1 + MD20 + RO33) was lower than that found in Myanmar (28.26% vs. 63.5%), but that single infection with K1 was higher (27.8% vs. 9.5%). Otherwise, single infection with MAD20 in Lao PDR was lower than in Myanmar (11.7% vs. 27%).
MOI has been suggested to differ in relation to age, transmission intensity and parasite density [31–33]. MOI in Lao PDR was rather low as not exceed 2.0, and was not correlated with age group like the previous report by Vafa et al. . The overall MOI in Lao PDR (1.6) was not different from the MOI reported in Vietnam (1.76) and Brazil (1.42), but lower than in Tanzania (2.37) and in Gabon (4.0) [22, 34]. There were many reports showed that the MOI was correlated with parasite density. MOI in this study did not increase with higher parasite density, similar to those reports in Brazzaville, Republic of Congo and in Gabon [22, 35]. This study used clinical P. falciparum isolates from patients with clinical symptoms, so that the MOI may be lower than that reported in asymptomatic children . Many reported showed that MOI would reflect that infected individual has immunity against malaria [32, 37]. Thus, the MOI should be studied in asymptomatic P. falciparum-infected population in Lao PDR further, especial in children.
The frequency of polymorphic alleles in the K1 allele type in Lao PDR and in Myanmar was not markedly different (12.34% vs. 9.5%), but the frequency of polymorphism in the MAD20 allelic type from Myanmar was much higher than that from Lao PDR (27% vs. 11.21%).
Lao PDR is a landlocked country with a long-space area. Ethnic minority populations are very localized distribution. Migration of the population within the country and cross-country is relatively low. Thus, the spread of polymorphic clones of msp-1 block2 allelic types of P. falciparum is limited to residents. Polymorphism of the K1, MAD20 and RO33 allelic types may occur in confined areas. Based on sequence data analysis and phylogenetic tree of each K1, MAD20 and RO33 allelic type sequences in Lao PDR with allelic type sequence data reported in other areas worldwide, found that Savannakhet, K1 type 6 and Savannakhet, RO33 type 2 may be unique polymorphic allelic types of P. falciparum-clinical isolate in Lao PDR.
Genetic polymorphism with diverse allelic types was identified in msp-1 block 2 in P. falciparum clinical isolates from Lao PDR. A rather high level of multiple clonal infections was also observed, but the degree of multiplicity of infection was rather low as not exceed 2.0. Clonal fluctuation in each allelic type was not observed. Most of the polymorphic sequences of each K1, MAD20 and RO33 allelic type were regional specific. This basic genetic data of msp-1 block 2 allelic types is useful for treatment and malaria control program in Lao PDR.
We are grateful to the patients who participated in the study, as well as their parents and guardians. We thank Human Research Development to Neighboring Countries for NK's scholarship, and the Graduate School, Khon Kaen University, for a generous research grant. Finally we thank Mr. Christopher Salisbury for suggestions and assistance with the English-language presentation of the manuscript.
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