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PCR genotyping: reliable or misleading?

November 28, 2016 - 17:04 -- Pierre Lutgen

A press release from Wellcome Trust Sanger Institute raised a lot of concern. Based on a scientific paper published in Nature

           Magnus Manske, Olivo Miotto, Susana Campino, Sarah Auburn, Jacob Almagro-Garcia, Gareth Maslen, Jack O’Brien, Abdoulaye Djimde, Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing. Nature 487, 375–379, 19 July 2012doi:10.1038/nature11174

Studies from 2009 already asked the same question

            Juliano JJ, Gadalla N, Sutherland CJ, Meshnick SR. The perils of PCR: can we accurately 'correct' antimalarial trials? Trends Parasitol. 2010 Mar;26(3):119-24. doi: 10.1016/j.pt.2009.12.007

            Jonathan J Juliano, Frederic Ariey, Rithy Sem, Noppadon Tangpukdee, Misclassification of Drug Failures in Plasmodium falciparum Clinical Trials in Southeast Asia. J Infect Dis. 2009 Aug 15; 200(4): 624–628. doi: 10.1086/600892

The use of genotyping to distinguish recrudescent from new infections is currently recommended for all clinical antimalarial efficacy trials by the World Health Organization. However, genotyping-adjusted drug efficacy estimates may vary between trials due to the use of different genotyping methods and to the different settings in which these methods are applied.

             William J Collins, Bryan Greenhouse, Philip J Rosenthal and Grant Dorsey. The use of genotyping in antimalarial clinical trials: a systematic review of published studies from 1995–2005. Malaria Journal 2006 5:122 DOI: 10.1186/1475-2875-5-122

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. Another recent publication confirms that a large number of submicroscopic P. falciparum infections were identified in a study area in Ethiopia. A significant number of individuals were asymptomatic, below the threshold of microscopy and RDT,

              Lemu Golassa, Nizar Enweji, Berhanu Erko, Abraham Aseffa and Göte Swedberg. Detection of a substantial number of sub-microscopic Plasmodium falciparum infections by polymerase chain reaction: a potential threat to malaria control and diagnosis in Ethiopia. Malaria Journal 201312:352 DOI: 10.1186/1475-2875-12-352

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.

               Curtis, C. F. & Otoo, L. N. A simple model of the build-up of resistance to mixtures of antimalarial drugs. 1986Trans. R. Soc.Trop. Med. Hyg. 80, 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.

               Druilhe P, Daubersies P, Patarapotikul J, Gentil C, Chene L, Chongsuphajaisiddhi T, Mellouk S, Langsley G. A primary malarial infection is composed of a very wide range of genetically diverse but related parasites. J Clin Invest. 1998 May 1;101(9):2008-16

                Ang HH, Chan KL, Mak JW. Clonal diversity in single isolates of Malaysian Plasmodium falciparum. Med Trop (Mars). 1996;56(4):349-51.

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.

                Cally Roper, Ibrahim M. Elhassan, Lars Hviid, Haider Giha, William Richardson, Hamza Babiker, Gwiria M. H. Satti, Thor G. Theander, and David E. Arnot . Detection of Very Low Level Plasmodium falciparum Infections using the Nested Polymerase Chain Reaction and a Reassessment of the Epidemiology of Unstable Malaria in Sudan. Am J Trop Med Hyg April 1996 54:325-331

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, A M Abdel-Muhsin, L C Ranford-Cartwright, G Satti and D Walliker Characteristics of Plasmodium falciparum parasites that survive the lengthy dry season in eastern Sudan where malaria transmission is markedly seasonal. Am J Trop Med Hyg October 1998 vol. 59 no. 4 582-590

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. A study run in Tanzania

            Anja M Carlsson, Billy E Ngasala, Sabina Dahlström, Christopher Membi, Isabel M Veiga, Lars Rombo, Salim Abdulla, Zul Premji, J Pedro Gil, Anders Björkman and Andreas Mårtensson.Plasmodium falciparum population dynamics during the early phase of anti-malarial drug treatment in Tanzanian children with acute uncomplicated malara. Malaria Journal201110:380. DOI: 10.1186/1475-2875-10-380

underlines the importance of interpreting PCR-outcomes with caution because they may overestimate assessment of drug efficacy in high transmission areas where genotypes are abundant.At the the 2d Nordic Malaria Conference at Copenhagen

            Dzikowski R, Deitsch KW Genetics of antigenic variation in Plasmodium falciparum. Curr Genet. 2009 Apr;55(2):103-10. doi: 10.1007/s00294-009-0233-2.

the genetic diversity of Plasmodium falciparum infections was confirmed 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.

            Cláudia Fançon, Miguel Brito and Jose Pedro Gil Plasmodium falciparum drug resistance in Angola. Malaria Journal201615:74. DOI: 10.1186/s12936-016-1122-z Other authors have raised questions about the validity of PCR genotyping

             Andreas Mårtensson, Billy Ngasala, Johan Ursing, M. Isabel Veiga, Lisa Wiklund, Christopher Membi, Scott M. Montgomery, Zul Premji, Anna Färnert and Anders Björkman. Influence of Consecutive-Day Blood Sampling on Polymerase Chain Reaction-Adjusted Parasitological Cure Rates in an Antimalarial-Drug Trial Conducted in Tanzania. 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

             Judy Natalia Jiménez, Carlos Enrique Muskus , Iván Darío Vélez . Diversidad genética de Plasmodium falciparum y sus implicaciones en la epidemiología de la malaria. 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. Most of the claims concerning the efficiency of ACTs are based on in vitro tests which predominantly evaluate the drug susceptibility on mature parasite stages, trophozoite or schizont. But the resistance to artemisinins may eventually be explained by a loss of susceptibility at the ring stage.

              Nicholas J. White Controversies in Tropical Medicine. Counter Perspective: Artemisinin Resistance: Facts, Fears, and Fables Am. J. Trop. Med. Hyg., 87(5), 2012, p. 785 doi:10.4269/ajtmh.2012.12-0573

In a literature survey on a large series of clinical trials.

               Mugittu K, Priotto G, Guthmann JP, Kiguli J, Adjuik M, Snounou G, Beck HP, Mshinda H, Olliaro PL, Taylor WR (2007) Molecular genotyping in a malaria treatment trial in Uganda - unexpected high rate of new infections within 2 weeks after treatment. Trop Med Int Health 12, 219-223. 2007

               Mugittu K, Genton B, Mshinda H, Beck HPMolecular monitoring of Plasmodium falciparum resistance to artemisinin in Tanzania. Malar J 19: 5, 126. (2006)

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: an accuracy of 70%. 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. The Four Artemisinin-Based Combinations (4ABC) Study Group. A Head-to-Head Comparison of Four Artemisinin-Based Combinations for Treating Uncomplicated Malaria in African Children: A Randomized Trial. PLoS Med 8(11): e1001119. doi:10.1371/journal.pmed.1001119

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.

               Anna Färnert, Marianne Lebbad, Lea Faraja and Ingegerd Rooth. Extensive dynamics of Plasmodium falciparum densities, stages and genotyping profiles Malaria Journal20087:241 DOI: 10.1186/1475-2875-7-241

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. Immunity also plays a key role in parasite clearance. A weak immune response will lead to slow clearance of parasites and the chances of new infections increase. Several papers from Gabon, Ghana, Senegal highlight this

                Buchholz U, Kobbe R, Danquah I, Zanger P, Reither K, Abruquah HH, Grobusch MP, Ziniel P, May J, Mockenhaupt FP. Multiplicity of Plasmodium falciparum infection following intermittent preventive treatment in infants. Malar J. 2010;9:244. doi: 10.1186/1475-2875-9-244

                Emmanuel Osei Tutu, John Larbi, Bernard Lawson, Genetic diversity of Plasmodium falciparum in pregnant women in an IPTp setting in the Offinso District ofGhana Journal of Parasitology and Vector Biology Vol. 3(1), pp. 12-18, January 2011

                Mombo LE, Ntoumi F, Bisseye C, Ossari S, Lu CY, Nagel RL, Krishnamoorthy R. Human genetic polymorphisms and asymptomatic Plasmodium falciparum malaria in Gabonese schoolchildren. AmJTrop Med Hyg. 2003 ;68: 186–190

                Jafari-Guemouri S, Boudin C, Fievet N, Ndiaye P, Deloron P. Plasmodium falciparum genotype population dynamics in asymptomatic children from Senegal. Microbes Infect. 2006;8: 1663–1670. doi: 10.1016/j.micinf.2006.01.023

Quoting the latter: “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.

                 Andrea Codd, Franka Teuscher, Dennis E Kyle, Qin Cheng and Michelle L Gatton Artemisinin-induced parasite dormancy: a plausible mechanism for treatment failure. Malaria Journal201110:56 DOI: 10.1186/1475-2875-10-56

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.

                Witkowski, B., Lelievre, J., Barragan, M. J., Laurent, V., Su, X. Z., Berry, A. and Benoit-Vical, F. (2010). Increased tolerance to artemisinin in Plasmodium falciparum is mediated by a quiescence mechanism. Antimicrobial Agents and Chemotherapy 54, 1872–1877. doi: AAC.01636- 09 [pii]; .

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.

               David D. Roberts, James A. Sherwood. Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence. Nature 318, 64 - 66 (07 November 1985); doi:10.1038/318064a0

The parasites which survive in the placenta or in the capillaries are different from those detected in the peripheral blood. 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 run in 2004 in RDCongo is used since 10 years as proof that Artemisia annua tea infusion doesn’t work. The results of this trial were not PCR corrected.

               Markus S. Mueller, Nyabuhanga Runyambo, Irmela Wagner, Steffen Borrmann, Klaus Dietze, Lutz Heide. Randomized controlled trial of a traditional preparation of Artemisia annua L. (Annual Wormwood) in the treatment of malaria Transactions of the Royal Society of Tropical Medicine and Hygiene(2004)98,318—321

ACT therapy does not measurably reduce human infectiousness to vectors in a setting of intense malaria transmission

              Bernadette J Huho, Gerard F Killeen, Heather M Ferguson, Adriana Tami, Christian Lengeler, J Derek Charlwood, Aniset Kihonda, Japhet Kihonda, S Patrick Kachur Thomas A Smith and Salim MK Abdulla. Artemisinin-based combination therapy does not measurably reduce human infectiousness to vectors in a setting of intense malaria transmission. Malaria Journal201211:118. DOI: 10.1186/1475-2875-11-118

Whilst it was disappointing for the authors, they conclude that it is not entirely surprising. Both rapid re-infection and semi-immune asymptomatic carriers are common in many African countries and infectious in settings of high transmission. PCR analysis after ACT treatment appears thus as a vain effort. 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.

               Sutherland CJ, lloueche A, Curtis J. Gambian children successfully treated with chloroquine can harbour and transmit Plasmodium falciparum gametocytes carrying resistance genes. Am J Trop Med Hyg 2002; 67:578-85

               Sutherland CJ, Drakeley CJ, Obisike U. The addition of artesunate to chloroquine for treatment of Plasmodium falciparum malaria in Gambian children delays, but does not prevent, treatment failure. Am J Trop Med Hyg 2003; 69:19-25

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, amino acids, unsaturated fatty acids, pentacyclic triterpenes, saponins and tannins. Artemisia annua is a true natural polytherapy.