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ABSTRACT |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Alternative markers that are easy to interpret and immunologically neutral are therefore required. Microsatellite markers, which are simple sequence repeats, are abundant in the genome of P. falciparum, occurring every 2–3 kb in both coding and non-coding sequences.6 These markers demonstrated a high degree of allelic variation and were used in studies of parasite population structure.7 However, they have never been applied in the context of a drug efficacy study for distinguishing recrudescence from new infection. We selected one polymorphic microsatellite of P. falciparum, a trinucleotide repeat (TAA)n located in the housekeeping gene polymerase alpha (Poly-
)7 and simplified its analysis through the use of capillary electrophoresis. The method was then applied to paired blood samples collected during a drug efficacy study and results were compared with those obtained by MSP typing.
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The amplification of the microsatellite (Poly) was done using the heminested polymerase chain reaction (PCR) conditions reported elsewhere.7 Primers PolyF, PolyR, and Poly-3(IR) were used,7 but none of them was end-labeled. Analysis of the PCR products was performed by capillary electrophoresis (Agilent 2100 Bioanalyser System; Agilent Technologies, Karlsruhe, Germany) in a microchip device (DNA 500, Labchip; Caliper Technologies, Mountain View, CA).
For PCR optimization and validation of the electrophoretic resolution, well-characterized sequence variants were necessary. Therefore, Poly PCR products of different size were identified in a polyclonal field sample and cloned using the TOPO cloning kit (Invitrogen, Carlsbad, CA). Plasmid DNA was extracted using a plasmid extraction kit (Wizard Plus minipreps; Promega, Madison, WI). Six clones showing size variation were sequenced using the dideoxy chain termination technique (GenBank accession numbers AJ851233, cl10; AJ851234, cl2; AJ851235, cl4; AJ851236, cl5; AJ851237, cl8; and AJ851238, cl9) to determine the precise size and sequence of the amplified Poly DNA fragment.
Statistical analysis was done using SPSS version 10.0.05 (SPSS, Inc., Chicago, IL). Children were considered not to be parasitological or clinical failures if their parasitemia between days 14 and 28 was classified as a new rather than recrudescent infection. Outcomes were defined according to the new World Health Organization classification10: clinical failure was defined as the sum of early treatment failure (ETF) and late clinical failure (LCF). Total treatment failure (TTF) includes parasitologic and clinical failure (ETF plus LCF plus late parasitologic failure). Comparison of outcomes between the two treatment groups was done using chi-square analysis.
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RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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For the interpretation of the microsatellite typing, two criteria were defined. First, to avoid counting an allele twice (as a result of the presence of primary PCR products), only fragments within a range of 49 basepairs above the smaller amplicon of a given sample were considered recrudescent (49 nucleotides is the difference between the position of the two reverse primers used in the hemi-nested reaction). Second, fragments with a size difference of less than three basepairs between paired samples were considered identical (MICRO-3 criterion). In most cases, several alleles were observed in each sample; if at least one allele was shared in the paired samples, this was interpreted as a recrudescence. A less stringent threshold was also evaluated: fragments with a size difference of less than four basepairs were considered identical (MICRO-4 criterion).
These criteria were applied to compare the degree of genetic diversity in the three sites. A higher number of microsatellite allelic types was found in Rukara (23 versus 19 in Kicukiro and 21 in Mashesha). For each marker and each setting, the ratio between the average number of allelic types observed in an individual at day 0 and the total number of alleles observed in all patients was computed. This provided for each marker an estimate of the probability of being reinfected with a specific allele. For Mashesha, the following results were observed: microsatellite markers (1.9/21 = 0.09), MSP-1 (3.1/14 = 0.22), and MSP-2 (4.1/20 = 0.20).
Of 308 children included in the drug efficacy study, 75 were parasitemic at either days 21 or 28 of follow-up.8 Sixty-nine paired samples could be analyzed by both the MSP (-1 and -2) and microsatellite markers. The latter (using both MICRO-3 and MICRO-4 criteria) showed more new infections (39/69 = 56.5% and 33/69 = 47.8%, respectively) than MSP (23/69 = 33.3%). Of 46 samples classified as recrudescence by MSP, 23 (50%) were classified as new infections by microsatellite markers. However, among the 30 samples classified as recrudescent by microsatellites markers, 7 (23.3%) were classified as new infections by MSP. No ETF was observed. Late clinical failure was higher in the AQ group than in the AQ plus AS group, and the difference became highly significant when corrected by the MSP PCR results.8 Similar results were obtained using MICRO-3 or MICRO-4. Total treatment failure was lower when corrected by the microsatellite markers than by MSP (Figure 1
). However, the relative risk for failure between AQ and AQ plus AS did not change substantially.
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). The majority (74%) of the other 23 patients classified as recrudescence by both MSP and microsatellite markers had more than five MSP alleles. Therefore, a more stringent MSP-based definition of recrudescence (at least one common allele in both MSP-1 and MSP-2 and at least five identical alleles in each pair instead of two) would decrease the discrepancy between MSP and microsatellite markers.
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) in distinguishing P. falciparum recrudescence from re-infection. The microsatellite technique has several advantages. The microsatellite PCR primers used are specific for P. falciparum and do not co-amplify other malaria species such as P. vivax and P. malariae.7 Our method of analysis is simpler than the one reported for population structure analysis where fluorescent end-labeled primers were used and the PCR product was analyzed with a sequencer.7 We used conventional, unlabeled primers and capillary electrophoresis for resolution of PCR products. Microsatellite genotyping is also faster to conduct than MSP typing (two versus six hours, respectively).
Analysis of field sample showed that based on microsatellite markers, 50% of the samples considered recrudescent by MSP markers were new infections. The new alleles detected by the microsatellites markers are unlikely to be due to the mutations during the testing period because the markers were stable over a one-year period. This should be checked for any new microsatellite marker used for typing, especially for those located in more unstable subtelomeric regions. The difference between microsatellite and MSP results is unlikely to be due to differences in mutation rates between the two markers. Indeed, microsatellites are considered to be rapidly evolving,11 but MSP genes can vary with a similar mechanism, i.e., amplification/deletion of short simple repeat DNA sequences.12 The difference could be explained by allelic diversity and the number of alleles per sample, which may influence the probability of re-infection with the same defined allele. When each marker was considered individually, this probability was smallest for the microsatellite markers (0.09 versus 0.22 and 0.20 for MSP-1 and MSP-2, respectively). However, when MSP-1 and MSP-2 were considered together, the probability, assuming complete independence between MSP-1 and MSP-2 (this happens only in a panmictic population), would be 0.044. Therefore, combining the two MSP markers should have a lower risk of misclassification. However, this is higher than expected when the two shared allele criterion (one for MSP-1 and one for MSP-2) are used. Indeed, 65.1% of discrepant cases shared 2–4 MSP alleles, while 74% of the congruent cases showed five or more shared MSP alleles (Table 1
). Finally, selective pressure could also be involved in the discrepancies between MSP and microsatellite markers. The latter are considered to be selectively neutral7 and prone to genetic drift, although possible selection by drug pressure was reported for microsatellite markers located around the drug target genes.13,14 In contrast, MSP are antigens and their variation is subject to diversifying natural selection because it contributes to the ability of the parasite to evade the immune response of the human host.12 Nevertheless, a fraction of infections classified as recrudescence by microsatellite were typed as new by MSP markers. Microsatellite homoplasy is considered to be rare,12 but this phenomenon might explain the latter results. Indeed, the Poly- region is organized into several blocks of microsatellites interrupted by few non-microsatellites bases.15 We found in our Poly- sequences that expansion of one block could be accompanied by contraction of another. If both variations would imply an identical number of repeats, the total length of the PCR-amplified product would remain the same. This problem could be overcome by using different microsatellites7,15 or by sequencing.
Under our experimental conditions, microsatellite markers showed more new infections than MSP. This suggests that the latter, when used in support to in vivo efficacy tests of one given drug, would overestimate failures. However, this apparently does not affect the comparison between different treatments. Indeed, in present case, even if the TTF varied for each of the two regimens, both MSP and microsatellite markers would show a lower TTF with AQ plus AS than with AQ.
Recrudescence is conceptually more complicated to demonstrate because until a difference between two paired samples has not been shown, a new infection cannot be excluded with certainty. A possible solution is to use both microsatellite and MSP markers together so that the probability of misclassifying new infection would be lower. In this case, since microsatellites are generally more resolvable than MSP, samples could first be analyzed by microsatellite markers; only those classified as recrudescence would be further tested by MSP-1 and MSP-2 markers for confirmation. Our results also suggest the need for re-evaluating the criteria used for interpreting MSP data by taking into consideration the diversity and frequency of allelic types in the parasite population under study. The use of more stringent criteria (more than two shared alleles) would be advisable.
Received November 22, 2004. Accepted for publication February 5, 2005.
Acknowledgments: We thank Dr. Claude Rwagacondo and Dr. Corinne Karema for collecting and supplying samples for our analysis, and Dr. G. Van Gemert (Koninklijke Universiteit van Nijmegen, Nijmegen, The Netherlands) for providing long-term cultivated P. falciparum DNA. We also thank the families in Rwanda for allowing samples to be obtained from their children.
Financial support: This study was supported by the Flemish Interuniversity Council, the East Africa Network for Monitoring Antimalarial Treatment, and the Belgian Development Co-operation.
* Address correspondence to Umberto D’Alessandro, Prince Leopold Institute of Tropical Medicine, Nationalstraat 155, B-2000 Antwerp, Belgium. E-mail: udalessandro{at}itg.be 
Authors’ address: Atunga Nyachieo, Chantal van Overmeir, Thierry Laurent, Jean-Claude Dujardin, and Umberto D’Alessandro, Department of Parasitology, Prince Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium, Telephone: 32-3-247-6355, Fax: 32-3-247-6359, E-mail: udalessandro{at}itg.be.
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REFERENCES |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
2, P < 0.05 (comparison between AQ and AQ + AS treatment groups).