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Is PCR genotyping of Plasmodium falciparum a reliable tool for monitoring drug resistance?

February 9, 2013 - 19:15 -- Pierre Lutgen

A recent press release from Wellcome Trust Sanger Institute raised a lot of concern. Based on a scientific paper published in Nature, 13 June 2012, it appears that a single infected person could harbour many genetically different Plasmodium falciparum parasites. The team from Oxford University found that these parasite populations easily swap DNA to create new forms.

This evidently raises the question how far PCR (polymer chain reaction genotyping) can distinguish between recrudescence (or treatment failure) and re-infection by new bites from anopheles mosquitoes.

In 1988, when studying the rate of build-up of resistance in a population where two anti-malarial drugs were used, either as a mixture or in sequence (CF Curtis et al., Trans R Soc Trop Med Hyg 80, 1988, 889-892), it was found that although resistances are initially rare, due to recombinations between the genes a large proportion of the parasite population remains unexposed to the 2 drugs.

In 1996, the Pasteur Institute in Paris found that a single mosquito inoculum would most likely be sufficient to generate a wide range of genetic diversity, even in the absence of repeated exposure. This diversity, which has been greatly underestimated in previous studies, does not bode well for the development of successful means of malaria control (P Druilhe et al., J Clin Invest, 101, 1998, 2008-16).

In 1996, also by using PCR to assay for low level Plasmodium falciparum infections that were below the threshold of detection of blood film examination, a Sudanese research group (C. Roper et al., Am J Trop Med Hyg 54, 1996, 325-331) found a substantial group of asymptomatic, submicroscopically patent infections within the population of a Sudanese village present throughout the year although clinical malaria episodes were almost entirely confined to the transmission season. The reservoir of parasite population was thus larger and more stable than previously thought, a finding that is consistent with the high levels of genetic variations at polymorphic loci reported from analysis of P. falciparum parasites in this area.

In 1998, a study on the characteristics of Plasmodium falciparum parasites that survive the lengthy dry season in an isolated village in Eastern Sudan (H A Babiker et al Am J Trop Med Hyg, 59, 1998, 582-590) shows that for people with asymptomatic infections some appear to harbour genetically complex infections, while others retain single clonal infections for several months. 21 of 26 patients with persisting parasitemias had initial infections containing multiple genotypes. Combinations of different alleles occur over time, even during the dry season. The authors verified that no mosquitoes could be caught during the months of April to June in this village. In the absence of mosquito transmission, the newly appearing clones could only have originated from parasites already present in the patients.

More recently at the the 2d Nordic Malaria Conference at Copenhagen, Sept 13-14 2012, R Dzikowski confirmed the genetic diversity of Plasmodium falciparum infections and the repertoires of multi-copy, hypervariable gene families. Via switching between diverse genes within these large families parasites have the capacity for rapid variation in the course of an infection. Other authors have raised questions about the validity of PCR genotyping (A. Martensson et al., J Infect Diseases, 195, 2007, 597-601) and state that because of the inherent constraints imposed by the biology of the parasite, PCR adjusted outcomes should be interpreted with caution to establish parasitological cure. Interpretation will significantly depend on methodology and this has critical implications for in vivo studies of anti-malarial drugs, especially in areas of high endemicity where genetic diversity of the parasite can be exponential.

An extensive review on the genetic diversity of Plasmodium falciparum and its implications in the epidemiology of malaria was published by the PECET in Medellin, Colombia( Biomédica, 25-4, 2005). This paper describes recombination events of the malaria parasite life cycle and their implications on the development of control measures in regions with different degrees of endemicity. The genetic diversity of Plasmodium falciparum is to a large extent responsible for the survival of this parasite, its capacity to evade the immune responses of the host as well as to produce strains resistant to drugs and vaccines.

The problem with artemisinin is that nobody knows exactly how it works or how resistance occurs at genetic level. Currently, genetic change in pfatpase6 gene is used to screen for resistance because pfatpase6 enzyme has been shown to be one of the targets for artemisinin; but it is also known that artemisinin has multiple targets. So, just basing resistance monitoring on one target for a drug with multiple targets makes deployment of PCR less reliable, although the use of multiple targets could make PCR a more reliable tool. PCR has also a much lower limit of detection than microscopy and is highly variable in sensitivity below a certain threshold. The major drawback of PCR however is high cost of reagents, equipment and requirement for special training (PE Engeu, personal communication)

In a literature survey on a large series of clinical trials (K. Mugittu et al., Trop Med & Internat Health 11, 2006, 1350-1359), it was found that of 3455 patients, 767 had post-day 14 recurrent parasitemia of which 686 could be genotyped: 246 were due to recrudescences, 286 due to new infections and 154 were unresolved. The latter represents a very high percentage of unresolved cases. In another head-to-head comparison of ACT’s and funded by the Institute of Tropical Medicine, Antwerp, Belgium and Sanofi-Aventis 4 116 children in 7 African countries were involved . Coartem was evaluated on 1 226 patients. At day 28 the efficacy was 72.7 % but PCR adjustment raised it to 95.5 %). The number of adverse events reported in this group was 61.9% (U d’Alessandro et al., PLoS Medicine,8:11, Nov 2011)

By assessing the dynamics of asymptomatic Plasmodium falciparum infections at intervals of a few hours over 5 days in a single individual, it was found that parasite densities, maturation stages and genotyping profiles vary extensively (A. Färnert et al., Malaria Journal 2008 7:241). In total, nine clones were distinguished in this one individual over 5 days. The profiles differed by up to six alleles in samples collected only six hours apart. A single blood sample for genotyping a Plasmodium falciparum population is close to meaningless and does not allow to distinguish between recrudescence and re-infection.

A similar conclusion is reached in a study in Senegal (S. Jafari et al., Microbes and Infection 8, 2006, 1663-1670). “In areas where malaria is endemic, infected individuals generally harbor a mixture of genetically distinct Plasmodium falciparum parasite populations. For the first time, we studied temporal variations of blood parasite densities and circulating genotypes in asymptomatic Senegalese children, at time intervals as short as 4-12 h. Twenty-one Senegalese children, presenting with an asymptomatic P. falciparum infection, were sampled eight times within three days. Parasite density was assessed by thick blood smears, and all infecting genotypes were quantified by the fragment-analysis method. Parasite densities showed dramatic fluctuations up to a 1 to 1,000 ratio, with at least one peak of parasite density. Polyclonal infections were detected in all children, with a multiplicity of infection of 5.2-6.8 genotypes per child. A single sample never reflected the full complexity of the parasite populations hosted by a given individual. Genotypes with different behaviours were detected in all children, some genotypes undergoing major fluctuations, while others were highly stable during the follow-up. A single peripheral blood sampling does not reflect the total parasite load. Repeated sampling is needed to have a more detailed scheme of parasite population dynamics and a better knowledge of the true complexity of an infection”.

PCR may also be affected by the dormancy effect (A. Codd et al., Malaria Journal, 10:56, 2011). One of the side effects of the higher doses of artemisinin is this effect induced in plasmodium. The parasite encapsulates itself against the aggressive peroxide artesunate and reawakens at the end of the treatment. The same effect is called quiescence by a French research team (B. Witkowski et al., Antimcro Agents Chemother. Doi:10.1128/AAC.01636-09). Under a very high dose of artesunate, a Plasmodium falciparum ring-stage sub-population persists in culture and continues its cycle of development normally after drug removal. This may be one of the causes of resistance. This dormancy effect is similar, but probably not related to the sequestration and cytoadherence of falciparum parasitized erythrocytes. Sequestration of these erythrocytes in capillaries or placenta may protect the parasite from destruction and hide most of it from PCR analysis of a blood sample (D. Roberts et al., Nature 318, 1985, 64-66). The parasites which survive in the placenta or in the capillaries are different from those detected in the peripheral blood (A. A. Alamin et al., Al Neelain Medical Journal 1, 2011, 1-9). After 14 days, they could indicate a false reinfection in PCR.

The WHO Guidelines for the Treatment of Malaria states: “The objective of treating uncomplicated malaria is to cure the infection as rapidly as possible,” with cure being defined as “the elimination from the body of the parasites that caused the illness.” Patient treatment regimens recommended in the WHO guidelines are those designed to achieve rapid and full elimination. But in their assessment of the efficiency of anti-malarial drugs WHO experts often do cherry-picking. All failures of ACT pills are corrected by PCR, but the clinical trial of M. Muller et al., (Elsevier 2004) is used since 10 years as proof that Artemisia annua tea infusion doesn’t work. The results of this trial were not PCR corrected.

This dogma often motivates the medical orthodoxy that patients should complete drug courses even when they no longer feel sick. But as A. Read et al. (PNAS, 1008, 2011, 10871-10877) state: “Yet radical pathogen cure maximizes the evolutionary advantage of any resistant pathogens that are present”. This is particularly the case for artemisinin derivates in ACTs. Artesunate stimulates the immune system at low doses and inhibits it at high doses (US Patent 5578637 H. Lai). The doses prescribed by WHO in ACTs are 100 times higher than the LD50. Wouldn’t it be preferable to give the immune system some initial help and then let it take over and deal with resistant parasites? Even in the absence of any clinical treatment, it can cure most of the malaria episodes in 10 days.

The associated aspect of the dogma that targets total parasite clearance by day 28 has been questioned recently by M L Willcox et al. (Trans Royal Soc Trop Med Hyg, 105, 2011, 23-31). In an area of high transmission, total clearance at day 28 was not correlated with incidence of uncomplicated or severe malaria over the subsequent two months.

Worrisome is also the recommendation made by WHO in September 2010 to use mass drug administration (MDA) as part of a strategy to contain artemisinin-resistant parasites in the Greater Mekong sub-region. MDA is the practice of treating a whole population within a given geographical area, irrespective of the presence of symptoms and without diagnostic testing. In general, WHO has discouraged MDA for routine malaria control because studies suggest that the impact on transmission is short-lived and because of the likelihood that it will result in selection for drug-resistant genotypes. There was always widespread agreement that stewardship of anti-malarials means restricting their use to only those patients who need them. Aggressive chemotherapy bears the risk of spread of resistance. This was one of the reasons why WHO over recent years has strongly recommended the use of rapid malaria detection tests before delivering any anti-malarial drug.

Intermittent preventive treatment has also been recommended (WHO/AFR/MAL/04/01), for children and even for pregnant women even in areas where 50% parasitological failure is reported on day 14. It has been demonstrated that it can reduce the incidence of clinical malaria. But there is serious concern over the widespread deployment of IPT and that this will enhance the spread of drug resistance. (A. Dicko et al, PLoS Medicine, doi/10.1371). Mutations associated with pyrimethamine and sulphadoxine resistance, respectively, were found significantly more frequently at the end of the malaria transmission season in parasites obtained from children treated by IPT. It was also found that in pregnant women the genetic diversity of Plasmodium falciparum is very high and this threatens the effectiveness of using sulphadoxine-pyrimethamine. (EO Tutu et al., J of Parasitol and Vector Biol. 3, 2011, 12-18). The increase in the frequency of these mutations and an increase in transmission by gametocytes have been reported by other research groups after the use of sulphadoxine-pyrimethamine (SP). A study in Mali however has shown that although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be low (A Beavogui et al., Inter J Parasitol., 40, 2010, 1213-20). On the other hand attractiveness of mosquitoes and their feeding behaviour on humans is related to the gametocyte load ( R Lacroix et al., PLoS Biol 3-9 2005 e298). An increase in transmission of gametocytes also has been reported for chloroquine (CQ), but in this case the gametocytes transmitted were significantly more prone to carry resistance genes (C. Sutherland et al., Am J Trop Med Hyg 67, 2002, 578). SP and CQ are thus likely to have spread a public health disaster over Africa by increasing malaria transmission

Confronted with all these conflicting instructions from WHO, what should the physician in Africa in areas of high malaria endemicity do?

As Lucy Kangethe from University at Nairobi stated in her MSc thesis in 2006 “The compounds in Artemisia annua could be more effective and cheaper in the treatment of malaria than the pure artemisinin and its derivates", probably because different combinations of these compounds have specific actions against specific genotypes of plasmodium. The plant contains a vast array of constituents with demonstrated anti-malarial properties: the families of essential oils, coumarins, flavonoids, polysaccharides, saponins and tannins.

Artemisia annua is a true natural polytherapy.

And it also has been demonstrated with large communities in Uganda and Kenya that Artemisia annua has excellent prophylactic properties against malaria.

Ahmed Hassanali, Kenyatta University

Pierre Lutgen, IFBV-Belherb

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