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Multiple-clone infections of Plasmodium vivax: definition of a panel of markers for molecular epidemiology.

de Souza AM, de Araújo FC, Fontes CJ, Carvalho LH, de Brito CF, de Sousa TN - Malar. J. (2015)

Bottom Line: Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones.These mixtures were generated by mixing cloned PCR products or patient-derived genomic DNA.The analysis of DNA mixtures showed that the tandem repeat MN21 and the polymorphic blocks 2 (msp1B2) and 10 (msp1B10) of merozoite surface protein-1 allowed for the estimation of the expected ratio of both alleles in the majority of preparations.

View Article: PubMed Central - PubMed

Affiliation: Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz (FIOCRUZ), Belo Horizonte, Minas Gerais, Brazil. aracelesouza@cpqrr.fiocruz.br.

ABSTRACT

Background: Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones. The detection of multiple-clone infections depends on several factors, such as the accuracy of the genotyping method, and the type and number of the molecular markers analysed. Characterizing the multiplicity of infection has broad implications that range from population genetic studies of the parasite to malaria treatment and control. This study compared and evaluated the efficiency of neutral and non-neutral markers that are widely used in studies of molecular epidemiology to detect the multiplicity of P. vivax infection.

Methods: The performance of six markers was evaluated using 11 mixtures of DNA with well-defined proportions of two different parasite genotypes for each marker. These mixtures were generated by mixing cloned PCR products or patient-derived genomic DNA. In addition, 51 samples of natural infections from the Brazil were genotyped for all markers. The PCR-capillary electrophoresis-based method was used to permit direct comparisons among the markers. The criteria for differentiating minor peaks from artifacts were also evaluated.

Results: The analysis of DNA mixtures showed that the tandem repeat MN21 and the polymorphic blocks 2 (msp1B2) and 10 (msp1B10) of merozoite surface protein-1 allowed for the estimation of the expected ratio of both alleles in the majority of preparations. Nevertheless, msp1B2 was not able to detect the majority of multiple-clone infections in field samples; it identified only 6 % of these infections. The merozoite surface protein-3 alpha and microsatellites (PvMS6 and PvMS7) did not accurately estimate the relative clonal proportions in artificial mixtures, but the microsatellites performed well in detecting natural multiple-clone infections. Notably, the use of a less stringent criterion to score rare alleles significantly increased the sensitivity of the detection of multi-clonal infections.

Conclusions: Depending on the type of marker used, a considerable amplification bias was observed, which may have serious implications for the characterization of the complexity of a P. vivax infection. Based on the performance of markers in artificial mixtures of DNA and natural infections, a minimum panel of four genetic markers (PvMS6, PvMS7, MN21, and msp1B10) was defined, and these markers are highly informative regarding the genetic variability of P. vivax populations.

No MeSH data available.


Related in: MedlinePlus

Expected and normalized observed ratios of molecular marker alleles amplified from mixtures of cloned DNA with predominance of A-type allele (a) and B-type allele (b). Two microsatellite markers (PvMs6 and PvMS7), one tandem repeat (MN21), and three antigen-coding markers (msp1B2, msp1B10 and msp3α) were PCR amplified and analysed by capillary electrophoresis. The dotted line indicates the expected ratios of peak heights according to the proportion of molecules (cloned DNA) from each allele used as a template for PCR amplification. Data represent the average of two independent experiments
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Fig1: Expected and normalized observed ratios of molecular marker alleles amplified from mixtures of cloned DNA with predominance of A-type allele (a) and B-type allele (b). Two microsatellite markers (PvMs6 and PvMS7), one tandem repeat (MN21), and three antigen-coding markers (msp1B2, msp1B10 and msp3α) were PCR amplified and analysed by capillary electrophoresis. The dotted line indicates the expected ratios of peak heights according to the proportion of molecules (cloned DNA) from each allele used as a template for PCR amplification. Data represent the average of two independent experiments

Mentions: Only three of the six markers (msp1B2, msp1B10 and the tandem repeat MN21) were able to detect the correct proportions of each clone in most of the artificial mixtures (Fig. 1 and Additional file 2). For both msp1 markers, the correspondence between the expected and observed proportions was evident, even for non-normalized data (Additional file 2). Thus, six to seven of ten tested dilutions corresponded to the expected proportions of the two cloned msp1 markers in the infection (Fisher´s exact test, P > 0.05). These results agree with linear regression analyses, which indicated a significant relationship between the actual peak height and the known mixture of pDNA based on high regression values (R2) and a slope close to the expected value of unity for both msp1 markers and MN21 (Additional file 2).Fig. 1


Multiple-clone infections of Plasmodium vivax: definition of a panel of markers for molecular epidemiology.

de Souza AM, de Araújo FC, Fontes CJ, Carvalho LH, de Brito CF, de Sousa TN - Malar. J. (2015)

Expected and normalized observed ratios of molecular marker alleles amplified from mixtures of cloned DNA with predominance of A-type allele (a) and B-type allele (b). Two microsatellite markers (PvMs6 and PvMS7), one tandem repeat (MN21), and three antigen-coding markers (msp1B2, msp1B10 and msp3α) were PCR amplified and analysed by capillary electrophoresis. The dotted line indicates the expected ratios of peak heights according to the proportion of molecules (cloned DNA) from each allele used as a template for PCR amplification. Data represent the average of two independent experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4548710&req=5

Fig1: Expected and normalized observed ratios of molecular marker alleles amplified from mixtures of cloned DNA with predominance of A-type allele (a) and B-type allele (b). Two microsatellite markers (PvMs6 and PvMS7), one tandem repeat (MN21), and three antigen-coding markers (msp1B2, msp1B10 and msp3α) were PCR amplified and analysed by capillary electrophoresis. The dotted line indicates the expected ratios of peak heights according to the proportion of molecules (cloned DNA) from each allele used as a template for PCR amplification. Data represent the average of two independent experiments
Mentions: Only three of the six markers (msp1B2, msp1B10 and the tandem repeat MN21) were able to detect the correct proportions of each clone in most of the artificial mixtures (Fig. 1 and Additional file 2). For both msp1 markers, the correspondence between the expected and observed proportions was evident, even for non-normalized data (Additional file 2). Thus, six to seven of ten tested dilutions corresponded to the expected proportions of the two cloned msp1 markers in the infection (Fisher´s exact test, P > 0.05). These results agree with linear regression analyses, which indicated a significant relationship between the actual peak height and the known mixture of pDNA based on high regression values (R2) and a slope close to the expected value of unity for both msp1 markers and MN21 (Additional file 2).Fig. 1

Bottom Line: Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones.These mixtures were generated by mixing cloned PCR products or patient-derived genomic DNA.The analysis of DNA mixtures showed that the tandem repeat MN21 and the polymorphic blocks 2 (msp1B2) and 10 (msp1B10) of merozoite surface protein-1 allowed for the estimation of the expected ratio of both alleles in the majority of preparations.

View Article: PubMed Central - PubMed

Affiliation: Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz (FIOCRUZ), Belo Horizonte, Minas Gerais, Brazil. aracelesouza@cpqrr.fiocruz.br.

ABSTRACT

Background: Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones. The detection of multiple-clone infections depends on several factors, such as the accuracy of the genotyping method, and the type and number of the molecular markers analysed. Characterizing the multiplicity of infection has broad implications that range from population genetic studies of the parasite to malaria treatment and control. This study compared and evaluated the efficiency of neutral and non-neutral markers that are widely used in studies of molecular epidemiology to detect the multiplicity of P. vivax infection.

Methods: The performance of six markers was evaluated using 11 mixtures of DNA with well-defined proportions of two different parasite genotypes for each marker. These mixtures were generated by mixing cloned PCR products or patient-derived genomic DNA. In addition, 51 samples of natural infections from the Brazil were genotyped for all markers. The PCR-capillary electrophoresis-based method was used to permit direct comparisons among the markers. The criteria for differentiating minor peaks from artifacts were also evaluated.

Results: The analysis of DNA mixtures showed that the tandem repeat MN21 and the polymorphic blocks 2 (msp1B2) and 10 (msp1B10) of merozoite surface protein-1 allowed for the estimation of the expected ratio of both alleles in the majority of preparations. Nevertheless, msp1B2 was not able to detect the majority of multiple-clone infections in field samples; it identified only 6 % of these infections. The merozoite surface protein-3 alpha and microsatellites (PvMS6 and PvMS7) did not accurately estimate the relative clonal proportions in artificial mixtures, but the microsatellites performed well in detecting natural multiple-clone infections. Notably, the use of a less stringent criterion to score rare alleles significantly increased the sensitivity of the detection of multi-clonal infections.

Conclusions: Depending on the type of marker used, a considerable amplification bias was observed, which may have serious implications for the characterization of the complexity of a P. vivax infection. Based on the performance of markers in artificial mixtures of DNA and natural infections, a minimum panel of four genetic markers (PvMS6, PvMS7, MN21, and msp1B10) was defined, and these markers are highly informative regarding the genetic variability of P. vivax populations.

No MeSH data available.


Related in: MedlinePlus