Review

Mutation rate

Mutation rate and the efficacy of antimicrobial drug treatment

Philip J Gerrish and J Gerardo Garca-Lerma
Despite rapid progress in drug development, microbial infections in general are becoming increasingly difficult to treat as a result of the emergence of drug-resistant strains. In some cases, such as HIV-1, the early goal of eradicating infections with antimicrobial drugs is, for now, being replaced with the more pragmatic goal of controlling infections over long periods of time through a succession of transiently effective treatments. Because treatment efficacy is often incomplete, studying the degree of treatment efficacy has great relevance to clinical disease management. We derived a model describing the association between the mutation rate of the pathogen and the degree of treatment efficacy. We found that drug
Panel 1. Mullers ratchet A
Figure 1. Mullers ratchet due to drug treatment. Black spots represent pretreatment harmful mutations; blue spots represent harmful mutations that occur during treatment; green spots represent a compensatory mutation; red squares represent resistance alleles. (A) genomes of the microbial population before drug treatment; the fittest genotype carries no harmful mutations. (B) genomes of the persisting population immediately following initiation of treatment; the fittest genotype carries two harmful mutations. (C) genomes of the persisting population some time after initiation of treatment; the fittest genotype carries three harmful mutations and one compensatory mutation. The compensatory mutation (green spots) is responsible for the increased fitness of C.

treatment is most effective when the mutation rate of the pathogen is either very low or, perhaps counterintuitively, very high. We discuss this finding in the light of a promising new treatment strategy for RNA viruses that combines antiviral compounds with a mutagen. Lancet Infect Dis 2003; 3: 2832

A principal determinant of treatment efficacy in microbial infections is the viability or fitness of the microbial population that persists despite treatment.1,2 If the persisting population has low fitness, for example, then the infected individuals microbial load is likely to remain low for a long period of

Theory. Suppose for simplicity that every non-lethal harmful mutation confers a selective disadvantage of s. Let U denote the genomic non-lethal harmful mutation rate of the virus. Haigh5 and later Johnson6 predicted that members of a large population would accumulate a stationary Poisson-distributed number of harmful mutations with mean U/s. When treatment is started, the fittest member of the small persisting population will be that with the fewest harmful mutations (see figure 1). Because of natural selection, this fittest member will largely determine the fitness of the persisting population. The least number of harmful mutations in the sample imposed by the bottleneck, here denoted X, has probability density, gX(x)=[1P(x1]n [1P(x)]n, where n is the number of pre-existing resistant mutants, or sample size selected, and P(x) is the Poisson distribution function (cdf) with parameter U/s. We now refine this equation by considering the sample size itself to be a Poisson random variable with mean n. Some algebra yields gX(x)=exp{n(x,U/s)/(x)} exp{n(x+1,U/s)/(x+1), where ( ) and ( , ) are the gamma and incomplete gamma functions, respectively. If pre-existing resistant mutants PJG is at the Department of Applied Mathematics, Instituto Mexicano are principally due to spontaneous mutation in the infected individual, then del Petrleo, Colonia San Bartolo Atepehuacan, Distrito Federal, their numbers will be linearly related to the mutation rate, such that n=kU. Mxico and the Los Alamos National Laboratory, Los Alamos, NM, On the other hand, if pre-existing mutants are principally due to USA; JGG-L is at the HIV and Retrovirology Branch, Division of AIDS, transmission, then their numbers will be independent of mutation rate, STD, and TB Laboratory Research, National Center for Infectious such that n is constant. The fitness of the persisting population is given by Diseases, Centers for Disease Control, Atlanta, GA, USA. (1s)X , where X is distributed as gX(X) . The resulting fitness expectation Correspondence: Dr Philip J Gerrish, Programa de Investigacin en and variance (under the assumptions of constant n and n=kU) are plotted Matematicas Aplicadas y Computacin, Instituto Mexicano del against U in figure 2. Expected fitness is plotted against k in figure 3. Petrleo, Colonia San Bartolo Atepehuacan, Distrito Federal, 07730
I I I

Mxico. Tel +52 5530037545; fax +52 5530036277; email pgerrish@yahoo.com

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Mutation rate

Review
is tit us ma r sis i o v to cr s A rs ne ru nza ula i n v e e c 1 le s V- lio flu si rus Sp viru HI Po In Ve vi

Panel 2. Simulations
A viral population was simulated in which harmful mutations, beneficial mutations, resistance mutations and mutator alleles were generated stochastically. Each generation, every virion in the population produced a number of offspring drawn at random from a Poisson distribution whose mean was determined by the virions fitness. A Poisson-distributed number of each kind of mutation was assigned to each of a virions progeny. At time zero, the simulated population was homogeneous for fitness, and all fitness and mutation rate variation was acquired by de novo spontaneous mutation. After 200 generations, drug treatment was initiated in two different ways. First, for figure 2A, resistance mutations were not incorporated. Instead, the population experienced a severe bottleneck, mimicking the initiation of drug treatment, by randomly choosing a prescribed number of virions to found in the persisting population. The factor by which this bottleneck reduced fitness was reported as the relative fitness of the persisting population (heavy dots in figure 2A), and it did not incorporate the additional fitness reduction due to the direct cost of resistance. Second, for figure 5, resistance mutations were incorporated and were assigned a direct fitness cost. The direct fitness cost was necessary to keep resistance mutations at low frequencies in the pretreatment population, in accordance with observations of real viral populations. Drug treatment was initiated by reducing the population to only those virions that carried resistance mutations.

A 10 08 Relative fitness 06 04 02 0 B 2 Log fitness variance 4

ia er ct a B

10 6 Relative fitness 8 10 3 2 1 Log U 0 1 2 08 06

U=01 U=1

U=2 04 02 0 05 10 15 2 0 25 30 Log K U=3 U=5

Figure 2. Fitness of the persisting population relative to the fitness of the pretreatment population, as a function of genomic harmful mutation rate of the infecting microbe. The governing equation is derived in panel 1, with parameter s=01. (A) expected relative fitness; (B) log fitness variance. Dashed curve assumes that pre-existing resistant mutants are primarily due to transmission, modelled by using constant n=10. Solid curve assumes that pre-existing resistant mutants are primarily due to spontaneous mutation within each infected individual, modelled by setting n=kU, where k=100. The heavy dots represent stochastic simulation results (panel 2). Fitness effects for both harmful and beneficial mutations were drawn at random from an exponential distribution with mean 01. Each new mutation-rate allele increased mutation rate by a factor that was drawn from an exponential distribution with mean 01. Harmful mutation rate estimates for some RNA viruses12 are shown as reference points. (We reason that the small genome size and strong codon bias of RNA viruses yield a harmful mutation rate that is similar to the genomic mutation rate.)

Figure 3. Fitness of the persisting population relative to the pretreatment population, as a function of k (defined in the text).

time, and the individual will be seen to respond well to treatment. In general, the fitness of the persisting population is inversely related to treatment efficacy. Our fundamental assumption is that the persisting population emerges from a small number of resistant mutants that are present when drug treatment is started. These pre-existing resistant mutants may be due to de novo mutation in the infected individual, or due to transmission. The fitness of the persisting population is affected by sampling effects. As a result of natural selection, the fitness of a population is determined largely by its fittest member. It is a matter of simple probability that the fittest member of the small resistant subpopulation is on average less fit than the fittest member of the large pretreatment population. Thus, drug treatment generally reduces the fitness of a microbial population, despite the emergence of resistance.

Such fitness loss is due to harmful mutations that have occurred at random positions on the genome. This sort of indiscriminate damage is easily acquired, but the highly discriminate reversions necessary to undo the damage are very unlikely to occur. It is thus difficult for the persisting population to recuperate the fitness lost.3,4 This process of largely irreversible fitness reduction because of sampling is known as Mullers ratchet,5 illustrated in figure 1 and modelled quantitatively in panel 1. (Mullers ratchet was originally formulated for populations of constant size, but the same notion applies to bottlenecked populations, as is the case here.) Using RNA virus populations, it has been shown that the fitness reduction due to Mullers ratchet can be substantial.711 Figure 2 shows how fitness of the persisting population changes as a function of increasing genomic mutation rate of the infecting microbe. There are two opposing factors that determine this fitness. The first is that, as mutation rate increases, the number of pre-existing resistant mutants in the population will increase, thereby increasing the expected fitness of the persisting population. The second, and perhaps less obvious, factor is that, as mutation rate increases, the persisting population will carry an increasingly large number

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Review
A 140 120 Viral titre (per mL) 100 80 60 40 20 100 B 200 300 400 Without mutagen With mutagen Treatment starts Panel 3. Optimum use of a mutagen

Mutation rate

5 0 100 5 200 300 400

Log viral titre (per mL)

If a mutagen is administered in conjunction with antivirals, as we propose, the question arises of how best to use the mutagen to enhance treatment. Firstly, the mutagen must be administered some time before the start of antiviral treatment. This time must be sufficient to allow mutation-selection balance to be approached in the population. Secondly, there is the question of whether or not to discontinue the use of the mutagen after initiating antiviral treatment. There are arguments for and against discontinuing the mutagen. If the mutagen is discontinued soon after initiation of the antiviral treatment, this has the advantage of slowing compensatory evolution and thus maintaining viral suppression for a longer period of time. On the other hand, if the mutagen is continued during antiviral treatment, Mullers ratchet would continue to operate faster in the persisting population and may even further increase viral suppression over time. Some data21 suggest that continuing the mutagen at least for some time after initiation of antiviral treatment would be the better strategy. This suggestion is corroborated by our simulation results, which show that the strongly suppressive effects of mutagen-antiviral treatment can sometimes be considerably delayed due to slow mutator enrichment dynamics (panel 4, results not shown). A more complete investigation of this matter is needed, however, and we thus leave it as a topic for further study.

10 15 Time (days)

Figure 4. Viral load trajectories with and without mutagen. The deterministic model of viral dynamics is given by equation (8) in Nowak and colleagues,18 and appropriate parameters for their HIV model are also found there. The mutagen modelled here confers only a threefold increase in the viral mutation rate. The fitness of the persisting population is prescribed by our Mullers ratchet model and is implemented in the model by adjusting the effective viral burst size. Fitness is assumed to remain constant following initiation of treatment. While of questionable realism, this assumption might reasonably approximate a balance between two opposing factors: compensatory evolution tending to increase fitness, countered by the further accumulation of harmful mutations tending to decrease fitness. After treatment begins, the green line indicates viral titres in the absence of a mutagen while the red line indicates viral titres in the presence a mutagen. (A) arithmetic scale, (B) log scale.

microbes and different drugs. Thus, the fitness of the persisting population could conceivably be significantly lower than our calculations suggest. Strictly speaking, therefore, our analytical calculations should be regarded as giving an upper bound on the relative fitness of the persisting population. Our simple quantitative analysis focuses on the initial fitness reduction as a result of drug treatment. There are strong arguments, and some data,2 to support the notion that the initial fitness reduction is directly correlated with not only the degree but also the duration of microbial suppression. During treatment, the infecting microbial population may eventually recuperate some or all of the fitness lost at the start of treatment. This ultimately results in the failure of that treatment to provide microbial suppression. The process of fitness recovery takes place through the reversion of harmful mutations and, more commonly, through the appearance of new mutations that compensate for the fitness lost
Panel 4. Mutator enrichment
Because of recurrent mutation in areas of the genome responsible for different aspects of replication and repair, mutation rate variants are present in most populations. Their frequency varies depending on the mutation rate of the organism in question and the proportion of its genome that is devoted to replication and repair. Furthermore, their frequency can be significantly higher than determined by a naive calculation of mutation-selection balance.6 For small RNA viruses, therefore, the frequency of mutator alleles (mutant genes that confer elevated mutation rates) in a population may be quite high. A selective bottleneck, such as that imposed on a microbial population by an antimicrobial drug, further increases the frequency of mutator alleles. This is because the particular trait being selected for (eg, resistance) is more likely to have been acquired on genomes that also carry a mutator allele. Work by Painter22 shows that a selective bottleneck increases the frequency of a mutator allele by a factor roughly equal to the factor by which the mutator allele increases mutation rate. In populations of RNA viruses where mutator alleles may already be at relatively high frequency, this effect may significantly elevate the population mutation rate. Such mutator enrichment has been reported in A cell populations23 and may explain reports of increased mutation rates in HIV immediately after in-vitro administration of antiretroviral drugs.24 Also, this event was reported in our simulations (figure 5). What this suggests is that, not only does a mutagen enhance the effect of an antiviral drug, but it is also the case that the selective bottleneck imposed by the antiviral drug can enhance the effect of the mutagen. As a result, Muller's ratchet should progress faster in the persisting population, further increasing treatment efficacy.

of harmful mutations, resulting in an increasingly non-viable persisting population. Our results (figure 2) show that this second factor can dominate when mutation rates are high. Figure 3 shows how fitness of the persisting population changes with increasing k. The parameter k is the factor that relates genomic harmful mutation rate, U, to the number of pre-existing resistant mutants, n, as n=kU. This relation comes from the finding that n and U are both linearly related to the total genomic mutation rate: n as a result of the balance between mutation and selection,13,14 and U as a result of the simple fact that some fraction of all mutations are harmful. Increasing k may loosely be interpreted as increasing pretreatment population size (ie, increasing microbial load), or decreasing fitness cost of resistance. Figure 3 shows that for high mutation rates, increasing k has little effect on the fitness of the persisting population. Our analytical model (panel 1 and figures 2, 3, and 4) ignores the direct fitness cost associated with carrying resistance to an antimicrobial drug. We have omitted this additional cost because it will be different for different

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Mutation rate

Review
B High genomic mutation rate

explanation for the limited success of drugs designed to treat RNA viral infections. Last, it suggests that if the 10 14 10 14 mutation rates of RNA viruses could be increased (using virus-specific 05 12 05 12 mutagens11,19), then antiviral treatment 0 0 0 0 efficacy could be improved. 1 51 101 151 201 251 1 51 101 151 201 251 Such a treatment strategy has recently been proposed and shown to work in vitro. Pariente and colleagues20 15 10 000 15 10 000 have shown that combinations of 1000 1000 10 10 antivirals together with a mutagen (to 100 100 increase mutation rate) can drive fit, 05 05 10 10 in-vitro populations of foot-and1 0 1 0 mouth disease virus to extinction. Our 1 51 101 151 201 251 1 51 101 151 201 251 results support this promising new Figure 5. Typical trajectories from stochastic simulations of viral populations, as described in panel 2. strategy (figures 4 and 5), they suggest Treatment was started at generation 201. Population carrying capacity was 11 000. Harmful mutations ways to refine it (see panel 3), and they occurred at a rate equal to the genomic mutation rate, and beneficial mutations occurred at a rate equal elucidate why it ought to work. The to 003 times the genomic mutation rate. Harmful and beneficial mutations had fitness effects drawn at validity of the proposed strategy is random from an exponential distribution with mean 01. Resistance mutations occurred at a rate equal further supported by a theoretical to 0003 times the genomic mutation rate, and resistance carried a direct fitness cost of 30%. (The total fitness drop at time of treatment in (A) is less than 30% because many resistant mutants had already investigation of error thresholds in acquired mutations compensating for this fitness cost.) Mutator mutations occurred at a rate equal to finite populations. Nowak and 01 times the genomic mutation rate, and they increased the genomic mutation rate by a factor drawn at Schuster18 reported that the maximum random from an exponential distribution with mean 01. (A) virus had an intermediate genomic mutation mutation rate tolerated by a rate of 005 mutations per replication. In this case, fitness recovery and viral rebound occurred. (B) virus had a higher mutation rate of 05 mutations per replication. Here, sustained fitness loss and viral population increases with increasing suppression were achieved. The sharp peak in mutation rate following initiation of treatment is the result population size. This finding of mutator enrichment and likely has a key role in driving down viral titre. Extinction was artificially qualitatively supports our findings that prevented in the simulations by disallowing less than one individual. the mutagen-enhanced mutation rate (figure 1).16,17 Compensatory mutations occur at a lower rate of a virus can be such that it is tolerated in the large in less fithence smallerpersisting populations, thus pretreatment population but not in the small post-treatment slowing the process of fitness recovery. In addition to having population (figure 5). slower compensatory evolution, smaller persisting We argue that the actions of mutagen and antiviral(s) populations accumulate further harmful mutations at a faster conveniently complement one another to enhance treatment rate, through the continued action of Mullers ratchet in its efficacy. As shown in figures 2 and 3, the increased load of traditional sense.5,18 Indeed, if the persisting population is harmful mutations provided by the mutagen decreases the small enough, it may continue to lose fitness gradually over expected fitness of the persisting population. Thus, the time as a result of Mullers ratchet, thereby increasing the mutagen enhances the viral suppression conferred by the duration of microbial suppression. The results of our antiviral(s). In panel 4, we argue that the bottleneck imposed simulations (panel 2, and figure 5) suggest that sustained by the antiviral(s) should further increase the mutation rate of fitness loss and microbial suppression are likely to be achieved the virus through mutator enrichment. This event was when pathogen mutation rate is high and/or the direct fitness reported in our simulation results (figure 5), and its effect cost of resistance is large. grows with increasing direct fitness cost of resistance. The Figure 2A shows that treatment success is most likely suppressive effect of the direct cost of resistance is therefore when the pathogen mutation rate is either very low or very amplified through its complementary effect on both Mullers high. First, this result suggests that the enormous ratchet and mutator enrichment. Thus, the antiviral(s) should effectiveness of antibiotics was due in part to the low also enhance the mutagen effect. In addition to this enhancing genomic mutation rates of bacteria. Second, comparison of synergy between mutagen and antiviral(s), the sharply the dashed and solid curves suggests that the present decreasing variance in fitness as U>1 increases (figure 2B) deterioration of antibiotic effectiveness is due largely to suggests that not only will a mutagen increase treatment transmission of resistant strains. Third, the figure offers an efficacy but, perhaps paradoxically, the increased error rate it bestows will also increase the certainty of effective treatment.
15 16 15 16

A Intermediate genomic mutation rate

Search strategy and selection criteria

Data for this review were identified by searches of BIOSIS, and references from relevant articles; numerous articles were identified through searches of the extensive files of the authors. Search terms were Mullers ratchet, error threshold, mutation rate, evolution of resistance, population bottlenecks, and antiviral therapy. English language papers were reviewed.
Acknowledgments

We thank P Baccam and J Gerrish for comments. This work was supported in part by the director-funded postdoctoral fellowship programme at Los Alamos National Laboratory (PJG).
Conflicts of interest

There are no conflicts of interest

Resistance frequency

Population size

Relative mutation rate

Fitness

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Review
References
1 Ioannidis JPA, Havlir DV, Tebas P, Hirsch MS, Collier AC, Richman DD. Dynamics of HIV-1 viral load rebound among patients with previous suppression of viral replication. AIDS 2000; 14: 148188. Quinones-Mateu ME, Ball SC, Marozsan AJ, et al. A dual infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression. J Virol 2000; 74: 922233. Muller HJ. Some genetic aspects of sex. Am Nat 1932; 8: 11838. Muller HJ. Evolution by mutation. Bull Am Math Soc 1958; 64: 13760. Haigh J. The accumulation of deleterious genes in a populationMullers ratchet. Theor Popul Biol 1978; 14: 25167. Johnson T. The approach to mutation-selection balance in an infinite asexual population, and the evolution of mutation rates. Proc R Soc Lond B Biol Sci 1999; 266: 238997. Domingo E, Escarmis C, Seville N, et al. Basic concepts in RNA virus evolution. FASEB J 1996; 10: 85964. Duarte E, Clarke D, Moya A, Domingo E, Holland J. Rapid fitness losses in mammalian RNA virus clones due to Mullers ratchet. Proc Natl Acad Sci USA 1992; 89: 601519. Duarte EA, Clarke DK, Moya A, Elena SF, Domingo 10 E, Holland J. Many-trillion-fold amplification of single RNA virus particles fails to overcome the Mullers ratchet effect. J Virol 1993; 67: 362023 Yuste E, Sanchez-Palomino S, Casado C, Domingo E, Lopez-Galindez C. Drastic fitness loss in human immunodeficiency virus type 1 upon serial bottleneck events. J Virol 1999; 73: 274551. Loeb LA, Essigmann JM, Kazazi F, Zhang J, Rose KD, Mullins JI. Lethal mutagenesis of HIV with mutagenic nucleoside analogs. Proc Natl Acad Sci USA 1999; 96: 149297. Drake JW, Charlesworth B, Charlesworth D, Crow JF. Rates of spontaneous mutation. Genetics 1998; 148: 166786. Haldane JBS. The mathematical theory of natural and artificial selection. Proc Camb Phil Soc 1927; 23: 83844. Gillespie JH. The Causes of Molecular Evolution. Oxford: Oxford University Press, 1991. Nowak MA, Bonhoeffer S, Shaw GM, May RM. Antiviral drug treatment: dynamics of resistance in free virus and infected cell populations. J Theor Biol 1997; 184: 20317. Levin BR, Perrot V, Walker N. Compensatory mutations, antibiotic resistance and the population genetics of adaptive evolution in bacteria. Genetics 2000; 154: 98597. Escarmis C, Davila M, Domingo E. Multiple molecular pathways for fitness recovery of an RNA virus debilitated by operation of Mullers ratchet.

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J Mol Biol 1999; 285: 495505. 18 Nowak M, Schuster P. Error thresholds of replication in finite populations mutation frequencies and the onset of Mullers ratchet. J Theor Biol 1989; 137: 37596. 19 Loeb LA, Mullins JI. Lethal mutagenesis of HIV by mutagenic ribonucleoside analogs. AIDS Res Hum Retroviruses 2000; 16: 13. 20 Pariente N, Sierra S, Lowenstein PR, Domingo E. Efficient virus extinction by combinations of a mutagen and antiviral inhibitors. J Virol 2001; 75: 972330. 21 Grande-Perez A, Sierra S, Castro MG, Domingo E, Lowenstein PR. Molecular indetermination in the transition to error catastrophe: systematic elimination of lymphocytic choriomeningitis virus through mutagenesis does not correlate linearly with large increases in mutant spectrum complexity. Proc Natl Acad Sci USA 2002; 99: 1293843. 22 Painter PR. Mutator genes and selection for the mutation rate in bacteria. Genetics 1975; 79: 64960. 23 Mao E, Lane L, Lee J, Miller J. Proliferation of mutators in A cell population. J Bacteriol 1997; 179: 41722. 24 Mansky LM, Bernard LC. 3-Azido-3deoxythymidine (AZT) and AZT-resistant reverse transcriptase can increase the in vivo mutation rate of human immunodeficiency virus type 1. J Virol 2000; 74: 953239.

Clinical picture
Diphyllobothriasis

A 54-year-old man was referred to hospital because of a positive occult blood in the mass-screening stool examination. Colonoscopy showed an actively moving tapeworm in the ascending colon to the ileocecal valve (figure). The patient had ingested a raw fillet of Oncorhynchus masou. The patient was treated with 300 mL of amidotrizoic acid through a duodenal tube. The tapeworm was seen descending from jejunum to the colon with peristalsis of the small intestine. The expelled worm was alive with intact scolex, and morphologically identical with Diphyllobothrium nihonkaiense. Tapeworm disease caused by eating raw beef and fish has shown a tendency to reoccur.
Uichiro Fuchizaki, Hideki Ohta, and Tatsuho Sugimoto.
UF is at the Department of Internal Medicine, Keiju Medical Center, Ishikawa, Japan; and HO and TS are at the Department of Gastroenterology, Tonami General Hospital, Toyama, Japan. Correspondence: Dr U Fuchizaki, Keiju Medical Center, 94 Tomioka-cho, Nanao, Ishikawa, 926-8605 Japan. Fax 81 767 52 7483; email fuchi@pl.coralnet.or.jp

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