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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Molecular genotyping is used increasingly to help distinguish recrudescence from reinfection in antimalarial drug efficacy studies.3 Infecting strains of malaria parasites can be "fingerprinted" through polymerase chain reaction (PCR) amplification of polymorphic genes.7 The "fingerprint" patterns of isolates causing successive episodes of malaria can then be compared to distinguish recrudescent from newly infecting parasites. Although the principle behind molecular genotyping is straightforward, the methods used to interpret results have varied widely. In particular, the definitions used to classify treatment failures as recrudescence or reinfection have not been consistent. This underestimation may be especially important when studying drugs with short half-lives, such as artesunate, and effective combination regimens, where a majority of treatment failures may occur after 14 days.
In the present study, we analyzed polymorphisms within the 3 genes—merozoite surface protein-1 (msp-1), merozoite surface protein-2 (msp-2), and glutamate-rich protein (glurp)—most commonly evaluated to distinguish recrudescence from reinfection in antimalarial drug efficacy studies. Our database was from a longitudinal comparison of 3 antimalarial regimens for the treatment of uncomplicated malaria in Kampala, Uganda. Statistical techniques were used to compare different definitions of recrudescence and reinfection and to evaluate the utility of results obtained with each gene and all 3 genes combined. We discuss the impact of different approaches and offer a simple genotyping system using msp-2 alone to type adequately infecting parasites from our study population.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sample selection and polymerase chain reaction genotyping. Blood was collected on filter paper (Whatman No. 3, Whatman Inc., Clifton, NJ) each time a new episode of malaria was diagnosed and when treatment failure occurred. Molecular genotyping was performed for all successive episodes of uncomplicated Plasmodium falciparum malaria treated with study drugs except that failures within 6 days of treatment were classified as recrudescence, episodes caused by P. falciparum followed by nonfalciparum malaria were classified as reinfections, and episodes after quinine therapy (for severe malaria or clinical failure within 14 days) were considered new infections.
Parasite DNA was extracted with chelex 100 Resin (Bio-Rad Laboratories, Hercules, CA) as previously described.8 The polymorphic regions of msp-1 (block 2), msp-2 (block 3), and glurp (R2 repeat region) were amplified by nested PCR. First-round PCR primers corresponded to conserved sequences flanking these regions.9 Second-round PCR primers were used to amplify the K1, MAD20, and RO33 allelic families of msp-110; the IC3D7 and FC27 allelic families of msp-211; and the repeat region of glurp.12 For controls, genomic DNA from HB3 and 3D7 laboratory strains was isolated by standard techniques.
Nested PCR products were analyzed by electrophoresis using 2% agarose for msp-1 and msp-2 and 1.5% agarose for glurp. Samples from an individual patient were run in adjacent lanes. If there was no amplification for any allelic family, PCR was repeated with 5 times the quantity of template DNA. If no amplification was detected after this second reaction, amplification was classified as unsuccessful. Gel images were digitized and molecular weights were assigned to bands using GelCompar II software (Applied Maths, Sint-Martens-Latem, Belgium). Densitometric curves were calculated for each gel lane, and bands with >2% of the density of the dominant band in each lane were assigned molecular weights.
Data interpretation. Genotyping patterns were compared for successive episodes of uncomplicated P. falciparum malaria. Based on analysis of the HB3 strain, alleles were considered the same if molecular weights were within 10 bp for msp-1 and msp-2 and within 20 bp for glurp. It was assumed that after a patient initially was treated for malaria, a subsequent episode was caused by either parasite strains present before treatment (e.g., recrudescence) or parasite strains acquired after treatment (e.g., reinfection). An outcome was defined as recrudescence if a subsequent sample contained identical alleles or a subset of the alleles present in the first sample. An outcome was defined as reinfection if a subsequent sample contained only new alleles. If a subsequent sample contained alleles present in the first sample and new alleles, the outcome initially was considered indeterminate.
Statistical analysis. Different criteria for classifying indeterminate outcomes were compared using a statistical modeling approach. Treatment regimen and pretreatment parasite density were identified as strong independent predictors of treatment outcome using a Cox proportional hazards model. Measures of association (hazard ratios) for these predictor variables first were estimated only for episodes in which the outcomes were defined clearly as recrudescence or reinfection. These parameter estimates were considered the reference standards. The analysis was repeated after including episodes with indeterminate outcomes, using varied classification criteria. The best method for classifying indeterminate outcomes was considered that in which the new parameter estimates were closest to our reference standards. In our comparisons of different genes and the application of genotyping to clinical results, the risks of recrudescence and reinfection were estimated using Kaplan-Meier survival analysis techniques. Survival functions were compared using the log-rank test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Genotyping was not performed if an episode was not preceded or followed by a study drug treatment (n = 59), subsequent treatment was <7 days after initial treatment (n = 23), the episode was followed by nonfalciparum malaria (n = 2), or the sample was missing (n = 4). PCR amplification was successful in 99%, 96%, and 98% of samples for msp-1, msp-2, and glurp and in 96% of samples when all 3 genes were combined. Of a possible 392 paired primary and subsequent samples, PCR results were available for 386, 371, 379, and 367 pairs for msp-1, msp-2, glurp, and when all 3 genes were combined.
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). msp-1 (45 alleles) and msp-2 (57 alleles) revealed considerably greater parasite diversity than glurp (28 alleles). Based on a simple multinomial probability equation, the likelihood of 2 random infections having identical alleles was 2% for msp-2, 5% for msp-1, and 7% for glurp. The mean number of alleles per sample (complexity of infection) also was greater with msp-1 (1.7) and msp-2 (1.7) than with glurp (1.4). Because of the lesser degree of polymorphism detected by glurp, it was not considered further as a single genetic marker.
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). Three classification schemes for indeterminate outcomes were considered: (1) all indeterminate outcomes considered recrudescence, (2) indeterminate outcomes considered reinfection if 
50% of the alleles in the subsequent sample were new (in this scheme, 90%, 91%, and 71% of episodes were classified as reinfections using results based on msp-1, msp-2, and all 3 genes), and (3) all indeterminate outcomes considered reinfections. Compared with our reference standards, parameter estimates changed considerably when indeterminate outcomes all were considered recrudescence (scheme 1). In contrast, parameter estimates were similar when all indeterminate outcomes were classified as reinfections (scheme 3) or most were classified as reinfections (scheme 2). Based on our prediction model, episodes with indeterminate outcomes were much more consistent with reinfection than recrudescence.
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). In contrast, survival functions based on msp-1 results showed significant differences between concordant and discordant outcomes, suggesting a less accurate estimate of true outcomes. For the same comparison between msp-2 and all 3 genes, outcomes were concordant in 89% (327 of 367) and discordant in only 11% (40 of 367) of comparisons, and survival functions for concordant and discordant data were not significantly different (Figure 3).
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). When comparing different classification schemes for indeterminate outcomes, scheme 2 provided the most robust results across the different genes (msp-1 alone, msp-2 alone, and all 3 genes combined). Compared with results based on msp-1 or msp-2 alone, the use of 3 genes combined showed the greatest variation in results across different indeterminate outcome classification schemes because of the larger number of indeterminate outcomes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have used results from a longitudinal study of antimalarial drug efficacy to compare methodologies critically and offer a standardized genotyping approach. In this study, we had a large sample size, extended follow-up covering multiple episodes of disease, and 3 different treatment groups covering a wide range of risk for recrudescence. We found that polymorphisms in either msp-1 or msp-2 were sufficiently diverse for us to distinguish adequately recrudescence from reinfection in our study population. In a direct comparison of analyses based on msp-1 and msp-2 polymorphisms, msp-2 provided a more accurate measure of treatment outcomes. Analyses considering polymorphisms in glurp and all 3 genes combined provided no additional utility. We propose a standard approach in which the analysis of msp-2 polymorphisms is used to distinguish recrudescence from reinfection.
Our genotyping results agreed with expected outcomes. Most recrudescences occurred within 28 days after treatment. The risk of recrudescence was highest in patients treated with SP monotherapy, the regimen that also was the least effective based on clinical and parasitologic criteria. Most reinfections occurred >28 days after treatment. The rate of reinfection was relatively constant after 42 days, when levels of all study drugs were expected to be low.18
Other investigators have suggested that in areas of high transmission, evaluation of recrudescence is achieved best by detailed analysis of a single, highly polymorphic locus.19 It also has been argued that the use of multiple markers may enhance the detection of diversity and decrease the chance of misclassifying new infections as recrudescence.3 In this study, we detected considerable parasite diversity with analyses of either msp-1 or msp-2, resulting in an acceptably low probability of outcome misclassification. When results from all 3 genes combined were used, the level of diversity increased but at the cost of a considerably larger proportion of outcomes being classified as indeterminate.
It was important to determine how best to classify outcomes initially classified as indeterminate. In these cases, patients developed recurrent malaria, and infecting parasites contained alleles present in the prior episode and new alleles. In our sample, the proportion of outcomes fitting this category was 24% using msp-1, 16% using msp-2, and 62% using all 3 genes. Approaches considered in previous studies have included omitting indeterminate outcomes from the analysis,20,21 considering them all as either recrudescences or reinfections,17,22,23 or other more complex methods.13,15 In many studies, it is unclear what method was used. In this study, we compared 3 different classification schemes, using as the reference standard the subset of outcomes that were definitive (either recrudescence, when subsequent isolates contained only alleles present in the prior episode, or reinfection, when a subsequent isolate contained only new alleles). Our analysis showed that the episodes initially classified as indeterminate were much more likely to be caused by reinfection than by recrudescence. Based on this result, we hypothesize that if residual and new parasite strains cocirculate, clinical symptoms are more likely to be caused by the new strain. This hypothesis is consistent with data from Gabon, in which the time to reappearance of parasites was similar in distribution when subsequent episodes contained only new alleles or when subsequent episodes contained new and old alleles.23
PCR-based genotyping is potentially limited by the sensitivity of the technique for detecting all allelic variants present in an infection.3 This can be especially problematic with low-level parasitemia or when one strain is a minor component of a multistrain infection.24 The problem of sensitivity for multiple alleles is relatively unimportant in drug efficacy studies, however, in which strains responsible for clinical illness are likely to be dominant and detected by PCR. Consistent with this conclusion, in our study 92% of our samples had a parasite density 2,000 asexual parasites/µl (96% 500 asexual parasites/µl), and geometric mean parasite density was not associated with the number of strains detected (P = 0.23).
The presence of gametocytes in blood samples can confound genotyping analyses.3 Outcomes can be misclassified as recrudescence if asexual parasites are cleared but gametocytes originating from the primary episode are detected at the time that a subsequent episode is diagnosed. Although the presence of gametocytes in subsequent samples was associated with recrudescence (P = 0.001), gametocytes occurred in only 16 of 371 paired samples (11 of these in the group treated with SP alone), suggesting that confounding as a result of gametocytemia was not a significant problem in our study.
Genotyping is an important tool for antimalarial drug efficacy studies, but its value has been limited by inconsistent methodology. We have shown that the results of our clinical trial varied considerably depending on the choice of genes for analysis and the interpretation of genotyping results. We have shown that in this high-transmission area, a single, highly polymorphic genetic marker adequately distinguished between recrudescence and reinfection. We support the analysis of msp-2 polymorphisms because this genetic marker provided more accurate results compared with msp-1 and equivalent results compared with all 3 genes combined. More studies are needed, however, to determine if msp-2 or another marker is the appropriate choice in other high-transmission areas. In low-endemicity regions, multiple genetic markers may be needed to detect sufficient parasite diversity. In addition, we recommend that when recurrent isolates contain old and new alleles, outcomes should be classified as reinfection. Our hope is that additional studies will allow the development of a simple, standardized approach to genotyping analyses in endemic areas of Africa. This approach should allow for more accurate estimation of treatment failure risk and improve comparability among studies.
Received July 26, 2002. Accepted for publication October 21, 2002.
Acknowledgments: We thank the clinical study team of B.M. Karakire, Marx Dongo, Sam Nsobya, Moses Kiggundu, Christopher Bongole, Regina Nakafero, Bridget K. Nzarubara, Pauline Byakika, and Sarah Kibirango; Sarah Staedke, for reviewing the manuscript; community leaders from the Kawempe Division of Kampala; and the study participants and their parents or guardians.
Financial support: This work was supported by the Fogarty International Center/National Institutes of Health (TW00007, TW01506, and AI43301), the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), and the Doris Duke Charitable Foundation Clinical Research Fellowship.
Authors’ addresses: Adithya Cattamanchi, Philip J. Rosenthal, and Grant Dorsey, Department of Medicine, San Francisco General Hospital, University of California, San Francisco, P.O. Box 0811, San Francisco, CA 94143, Telephone: 415-206-8687, Fax: 415-648-8425. Daniel Kyabayinze, Department of Biochemistry, Makerere University Medical School, P.O. Box 7072, Kampala, Uganda. Alan Hubbard, Department of Biostatistics, School of Public Health, University of California, Berkeley, 735 University Hall, Berkeley, CA 94720, Telephone: 510-642-8365, Fax: 510-642-5815.
Reprint requests: Grant Dorsey, Department of Medicine, San Francisco General Hospital, University of California, San Francisco, P.O. Box 0811, San Francisco, CA 94143, Telephone: 415-206-8687, Fax: 415-648-8425, E-mail: grantd{at}itsa.ucsf.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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