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Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar.

Juliano JJ, Randrianarivelojosia M, Ramarosandratana B, Ariey F, Mwapasa V, Meshnick SR - Malar. J. (2009)

Bottom Line: This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples.RFLP analysis failed to detect any pfcrt K76T-bearing isolates.The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of North Carolina, Chapel Hill, USA. jjuliano@med.unc.edu

ABSTRACT

Background: Strains of Plasmodium falciparum genetically resistant to chloroquine (CQ) due to the presence of pfcrt 76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant P. falciparum isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect pfcrt 76T-bearing P. falciparum minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings.

Methods: This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect pfcrt 76T-bearing minority variant P. falciparum. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples.

Results: Thirty one clinical P. falciparum isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the pfcrt K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant P. falciparum strains after therapy as detected by HTA. RFLP analysis failed to detect any pfcrt K76T-bearing isolates.

Conclusion: These findings indicate that genetically CQ-resistant P. falciparum are more common than previously thought in Madagascar even though the fitness of the minority variant pfcrt 76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the surveillance of anti-malarial resistance. The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries.

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Dynamic range of imaging modalities. Panel A shows the detected band intensities by autoradiography of a serial dilution of genomic DNA detected by the DIG-HTA. Intensities are expressed as the ratio of the sample's intensity divided by the intensity of the highest concentration sample in order to account for inter gel variability. The gels were exposed for 5 minutes (diamonds), 10 minutes (squares) and 15 minutes (triangles). Panel B shows the same dilution series detected by CCD camera at 10 minutes (squares) with the best fit trend line (solid black line). The 5 minute and 15 minute detections are not shown.
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Figure 2: Dynamic range of imaging modalities. Panel A shows the detected band intensities by autoradiography of a serial dilution of genomic DNA detected by the DIG-HTA. Intensities are expressed as the ratio of the sample's intensity divided by the intensity of the highest concentration sample in order to account for inter gel variability. The gels were exposed for 5 minutes (diamonds), 10 minutes (squares) and 15 minutes (triangles). Panel B shows the same dilution series detected by CCD camera at 10 minutes (squares) with the best fit trend line (solid black line). The 5 minute and 15 minute detections are not shown.

Mentions: The DIG-HTA was also able to reproducibly quantitate the relative amount of DNA in the mixtures of genomic DNA (Table 1). However, each of the detection methods has a different dynamic range of detection. Therefore, different concentrations of control P. falciparum DNA were analysed by each method to determine the dynamic range in which quantification remains linear. Figure 2 shows the relative intensity of the detected bands, expressed as a ratio of the individual band intensity over the intensity of the band from the highest concentration. This was done in order to normalize for variability in detection between the gels. Autoradiography (Figure 2, Panel A) has the narrowest dynamic range. The signal for 0.1 ng of genomic DNA becomes nearly saturated at 10 minutes or longer of exposure. However, at 5 minutes of exposure the lower concentrations of DNA are not easily detectable. This dynamic can potentially affect the ability to detect and quantify minority variants (Figure 3). CCD cameras and phosphorimagers have a larger dynamic range and detection remains linear over a larger range. Figure 2 Panel B shows a 10 minute exposure to a CCD camera of a dilution series of DNA detected by chemiluminescence. The relationship remains linear over the entire concentration range (R2 = 0.9832) and over different exposure times [5 minute exposure (R2 = 0.9629) and 15 minute exposure (R2 = 0.9679)].


Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar.

Juliano JJ, Randrianarivelojosia M, Ramarosandratana B, Ariey F, Mwapasa V, Meshnick SR - Malar. J. (2009)

Dynamic range of imaging modalities. Panel A shows the detected band intensities by autoradiography of a serial dilution of genomic DNA detected by the DIG-HTA. Intensities are expressed as the ratio of the sample's intensity divided by the intensity of the highest concentration sample in order to account for inter gel variability. The gels were exposed for 5 minutes (diamonds), 10 minutes (squares) and 15 minutes (triangles). Panel B shows the same dilution series detected by CCD camera at 10 minutes (squares) with the best fit trend line (solid black line). The 5 minute and 15 minute detections are not shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664821&req=5

Figure 2: Dynamic range of imaging modalities. Panel A shows the detected band intensities by autoradiography of a serial dilution of genomic DNA detected by the DIG-HTA. Intensities are expressed as the ratio of the sample's intensity divided by the intensity of the highest concentration sample in order to account for inter gel variability. The gels were exposed for 5 minutes (diamonds), 10 minutes (squares) and 15 minutes (triangles). Panel B shows the same dilution series detected by CCD camera at 10 minutes (squares) with the best fit trend line (solid black line). The 5 minute and 15 minute detections are not shown.
Mentions: The DIG-HTA was also able to reproducibly quantitate the relative amount of DNA in the mixtures of genomic DNA (Table 1). However, each of the detection methods has a different dynamic range of detection. Therefore, different concentrations of control P. falciparum DNA were analysed by each method to determine the dynamic range in which quantification remains linear. Figure 2 shows the relative intensity of the detected bands, expressed as a ratio of the individual band intensity over the intensity of the band from the highest concentration. This was done in order to normalize for variability in detection between the gels. Autoradiography (Figure 2, Panel A) has the narrowest dynamic range. The signal for 0.1 ng of genomic DNA becomes nearly saturated at 10 minutes or longer of exposure. However, at 5 minutes of exposure the lower concentrations of DNA are not easily detectable. This dynamic can potentially affect the ability to detect and quantify minority variants (Figure 3). CCD cameras and phosphorimagers have a larger dynamic range and detection remains linear over a larger range. Figure 2 Panel B shows a 10 minute exposure to a CCD camera of a dilution series of DNA detected by chemiluminescence. The relationship remains linear over the entire concentration range (R2 = 0.9832) and over different exposure times [5 minute exposure (R2 = 0.9629) and 15 minute exposure (R2 = 0.9679)].

Bottom Line: This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples.RFLP analysis failed to detect any pfcrt K76T-bearing isolates.The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of North Carolina, Chapel Hill, USA. jjuliano@med.unc.edu

ABSTRACT

Background: Strains of Plasmodium falciparum genetically resistant to chloroquine (CQ) due to the presence of pfcrt 76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant P. falciparum isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect pfcrt 76T-bearing P. falciparum minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings.

Methods: This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect pfcrt 76T-bearing minority variant P. falciparum. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples.

Results: Thirty one clinical P. falciparum isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the pfcrt K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant P. falciparum strains after therapy as detected by HTA. RFLP analysis failed to detect any pfcrt K76T-bearing isolates.

Conclusion: These findings indicate that genetically CQ-resistant P. falciparum are more common than previously thought in Madagascar even though the fitness of the minority variant pfcrt 76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the surveillance of anti-malarial resistance. The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries.

Show MeSH
Related in: MedlinePlus