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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.The PCR-capillary electrophoresis-based method was used to permit direct comparisons among the markers.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

Detection of alleles in artificial mixtures of plasmid DNA by applying different criteria for rare allele identification. The frequency of detection of multiple alleles was calculated considering all 11 of the artificial mixtures assayed for each marker. The bars represent the total proportion of infections identified by each marker. Two criteria for minor allele detection were considered: a cut-off value of one-third (colored in black only) or one-fourth (the entire bar, including both black and greybars), of the height of the predominant peak. The increase in the rate of detection of alleles with one-fourth criterion is highlighted in grey
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Fig2: Detection of alleles in artificial mixtures of plasmid DNA by applying different criteria for rare allele identification. The frequency of detection of multiple alleles was calculated considering all 11 of the artificial mixtures assayed for each marker. The bars represent the total proportion of infections identified by each marker. Two criteria for minor allele detection were considered: a cut-off value of one-third (colored in black only) or one-fourth (the entire bar, including both black and greybars), of the height of the predominant peak. The increase in the rate of detection of alleles with one-fourth criterion is highlighted in grey

Mentions: Two criteria are frequently used to score rare alleles in multiple-clone infections: (1) the one-third criterion, wherein peak heights of rare alleles are equal or higher than 33 % of the height of the predominant peak, and (2) the one-fourth criterion, in which a cut-off value of 25 % is applied to detect rare alleles. When the one-third criterion was applied, PvMS6 and both msp1 markers detected 36–45 % of multiple infections (Fig. 2). For PvMS6, the use of the one-fourth criterion allowed an increase of 18 % in the detection rate of multiple infections, increasing the overall rate to 54 %. For PvMS7, msp3α and MN21, the multi-clonal parasite rates were below 30 % using either of the cut-off value criteria (25 or 33 %).Fig. 2


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)

Detection of alleles in artificial mixtures of plasmid DNA by applying different criteria for rare allele identification. The frequency of detection of multiple alleles was calculated considering all 11 of the artificial mixtures assayed for each marker. The bars represent the total proportion of infections identified by each marker. Two criteria for minor allele detection were considered: a cut-off value of one-third (colored in black only) or one-fourth (the entire bar, including both black and greybars), of the height of the predominant peak. The increase in the rate of detection of alleles with one-fourth criterion is highlighted in grey
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Detection of alleles in artificial mixtures of plasmid DNA by applying different criteria for rare allele identification. The frequency of detection of multiple alleles was calculated considering all 11 of the artificial mixtures assayed for each marker. The bars represent the total proportion of infections identified by each marker. Two criteria for minor allele detection were considered: a cut-off value of one-third (colored in black only) or one-fourth (the entire bar, including both black and greybars), of the height of the predominant peak. The increase in the rate of detection of alleles with one-fourth criterion is highlighted in grey
Mentions: Two criteria are frequently used to score rare alleles in multiple-clone infections: (1) the one-third criterion, wherein peak heights of rare alleles are equal or higher than 33 % of the height of the predominant peak, and (2) the one-fourth criterion, in which a cut-off value of 25 % is applied to detect rare alleles. When the one-third criterion was applied, PvMS6 and both msp1 markers detected 36–45 % of multiple infections (Fig. 2). For PvMS6, the use of the one-fourth criterion allowed an increase of 18 % in the detection rate of multiple infections, increasing the overall rate to 54 %. For PvMS7, msp3α and MN21, the multi-clonal parasite rates were below 30 % using either of the cut-off value criteria (25 or 33 %).Fig. 2

Bottom Line: Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones.The PCR-capillary electrophoresis-based method was used to permit direct comparisons among the markers.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