scispace - formally typeset
Search or ask a question
Journal ArticleDOI

PCR correction strategies for malaria drug trials: updates and clarifications.

TL;DR: The aim is to update scientists from public and private bodies who are working on the development, deployment, and surveillance of new malaria drugs, and argue for the adoption of improved standardised PCR correction methodologies.
Abstract: Malaria drug trials conducted in endemic areas face a major challenge in their analysis because it is difficult to establish whether parasitaemia in blood samples collected after treatment indicate drug failure or a new infection acquired after treatment. It is therefore vital to reliably distinguish drug failures from new infections in order to obtain accurate estimates of drug failure rates. This distinction can be achieved for Plasmodium falciparum by comparing parasite genotypes obtained at the time of treatment (the baseline) and on the day of recurring parasitaemia. Such PCR correction is required to obtain accurate failure rates, even for new effective drugs. Despite the routine use of PCR correction in surveillance of drug resistance and in clinical drug trials, limitations inherent to the molecular genotyping methods have led some researchers to question the validity of current PCR correction strategies. Here we describe and discuss recent developments in these genotyping approaches, with a particular focus on method validation and limitations of the genotyping strategies. Our aim is to update scientists from public and private bodies who are working on the development, deployment, and surveillance of new malaria drugs. We aim to promote discussion around these issues and argue for the adoption of improved standardised PCR correction methodologies.

Summary (3 min read)

INTRODUCTION

  • Cure rates in clinical trials of antimalarial drugs are based on clinical assessments and microscopy conducted during several weeks of post-treatment follow-up.
  • These rates may be corrected after genotyping parasites in patient blood samples to distinguish recrudescent infections (i.e. those containing parasites that survived drug treatment) from new infections (NI) acquired after treatment.
  • The ‘PCR corrected efficacy’ excludes all NI from treatment failures and is essential when trials are conducted in high transmission areas with frequent NI because, in those areas, even a perfectly efficacious drug would have a high apparent failure rate as, without correction, NIs would be mistaken for drug failures.
  • PCR-corrected cure rates and adequate clinical and parasitological response (considered as primary endpoints by European Medicines Agency and World Health Organization (WHO) are now routinely reported as primary endpoints in regulatory trials of new drugs, yet the United States Food and Drug Administration (FDA) requires uncorrected cure rates as primary endpoints and FDA’s assessment of PCR-corrected rates is still pending.
  • The WHO widely implements ‘PCR-correction’ in surveillance of drug efficacy in malaria endemic areas and recommends changing first-line antimalarial therapy if the PCR-corrected failure rate exceeds 10%.

PRINCIPLE OF PCR-CORRECTION

  • High frequency of multi-clonal infections is a hallmark of Plasmodium falciparum epidemiology, whereby genetically distinct parasite clones persist concurrently and over long periods of time.
  • Patients in high-transmission areas harbour a mean of about five parasite clones, and one or two clones in regions of intermediate or low transmission.
  • In 2007 a group of experts, convened by WHO and the Medicines for Malaria Venture (MMV), released recommendations for genotyping protocols, and a consensus for the analysis of genotyping data and outcome classification was presented.
  • Recrudescence is defined by a genotype that had already been detected in the blood sample taken prior to treatment (i.e. alleles are shared at Day 0 and Day X at all three loci).
  • 6 LIMITATIONS INHERENT IN THE CURRENT, WHO/MMV-RECOMMENDED METHOD OF.

PCR-CORRECTION

  • The authors review limitations of ‘PCR-correction’ and summarize the impact of parasite biology on clone detectability.
  • These limitations are often insufficiently acknowledged by users and in publications.

A. Biological and epidemiological limitations

  • Biological constraints resulting in undetected clones Parasite clones occasionally remain undetected by PCR despite their presence in the host, an observation denoted as ‘imperfect clone detectability’.
  • The longer the follow-up period, the more likely NIs are acquired.
  • Even when three markers were analysed, extremes in both low and high transmission cause problems for PCR-correction.
  • Genotyping methods will suffer from lack of discriminatory power in areas with almost clonal parasite population structure.
  • The persistence of gametocytes alone was not considered a criterion for treatment failure.

B. Technical, genotyping limitations

  • Allelic suppression and detection limit of minority clones Systematic investigation of technical limitations has revealed that PCR template competition during amplification (allelic suppression) contributes to imperfect detectability of individual clones.
  • Such excellent sensitivity in detecting minority clones has not been reached by methods other than allele-specific PCR.
  • The sequential typing strategy recommended by WHO/MMV4 has some pitfalls because it recommends that no further markers be typed if one marker indicated the presence of a NI on Day X. Consequently, if genotyping of the first marker suffered from a technical shortfall and erroneously identified a NI, no additional typing result would question that outcome.
  • Glurp as first marker seems problematic because among all markers glurp suffered from greatest amplification bias.
  • In trial sites where MOI is very low, most infections will be single-clone and clone competition would be largely absent.

TO MINIMIZE THE IMPACT OF THESE LIMITATIONS

  • Maximized precision of fragment sizing Substantial efforts have been made to overcome the above limitations and improve genotyping methodology.
  • CE has excellent resolution and can be considered highly robust if carefully determined cut-offs eliminate stutter peaks.
  • A re-analysis of a larger clinical trial conducted in Rwanda by different analysis approaches using simulation models16 suggested that these methods differ two-fold in how frequently they identify recrudescences (Jones and Hasting, personal communication).
  • A Bayesian algorithm was developed to adjust drug efficacy for length of microsatellite PCR products and population frequency of each genotype detected in paired samples,28 permitting to estimate the probability of misclassification and allelic suppression.
  • This highly promising new approach incorporates the uncertainty around the classification of NI and recrudescence.

POSSIBLE USE OF ALTERNATIVE GENOTYPING TECHNIQUES

  • This section briefly describes developments with great potential to improve genotyping of P. falciparum multiple-clone infections.
  • One suggestion is to identify regions of <500 bp that are 9 rich in single nucleotide polymorphisms (SNP), which can be genotyped by next generation sequencing (NGS).29-33 SNP-based typing methods have improved ability to detect minority clones: Low-abundant P. falciparum clones can be detected at a ratio of 1:1000 in mixed infections.
  • 29,31,32 Molecular inversion probes may be useful for highly multiplexed targeted sequencing33.
  • Thus, these should be validated in clinical trials at high priority.
  • Yet, biological constraints will remain the same, such as sequestration or detection limits for minority clones.

STEPS FORWARD AND CONCLUSIONS

  • The community of technical experts, trial investigators, regulators and policy makers should consider rapid adoption of new consensual protocols for genotyping in regulatory malaria drug trials, with harmonized laboratory procedures and data analyses.
  • In particular, obtaining FDA validation of PCR-correction in drug efficacy trials requires a demonstrably robust genotyping strategy generating reproducible results.
  • While the intrinsic biological constraints from the parasite’s biology cannot be resolved, all technical issues should be addressed jointly by laboratories involved in recrudescence typing.
  • For critical discussion and sharing their knowledge the authors wish to thank other experts in the field who supported their work.
  • IH validated genotyping approaches by modelling, contributed figure 1, and edited the article.

Did you find this useful? Give us your feedback

Content maybe subject to copyright    Report

Elsevier Editorial System(tm) for The Lancet
Infectious Diseases
Manuscript Draft
Manuscript Number: THELANCETID-D-18-01280R1
Title: PCR-correction strategies for malaria drug trials: Updates and
Clarifications
Article Type: Personal View
Corresponding Author: Dr. Ingrid Felger,
Corresponding Author's Institution:
First Author: Ingrid Felger
Order of Authors: Ingrid Felger; Georges Snounou, Dr.; Ian Hastings, Dr.;
Joerg J Moehrle, Dr.; Hans-Peter Beck, Prof. Dr.
Abstract: Malaria drug trials conducted in endemic areas face a major
challenge in their analysis: parasitaemia in blood samples collected
after treatment may indicate a drug failure or originate from a new
infection (NI) acquired after treatment. It is therefore vital to
reliably distinguish drug failures from NI to obtain accurate estimates
of drug failure rates. This is achieved in Plasmodium falciparum by
comparing parasite genotypes obtained at time of treatment (the baseline)
and on the day of recurring parasitaemia. Such 'PCR-correction' is
required, even for new effective drugs, to obtain accurate failure rates.
Despite its routine use in surveillance of drug resistance and in
clinical drug trials, limitations inherent to the molecular genotyping
methods has led some commentators to question the validity of current
PCR-correction strategies. We describe and discuss recent developments in
these genotyping strategies with particular focus on method validation
and limitations. Our aim is to update scientists from public and private
bodies working towards the development, deployment and surveillance of
new malaria drugs. We aim to promote discussion around these issues and
argue for the adoption of improved standardized PCR-correction
methodologies.

1
Dear Editor
Thank you for sending us the reviewers comments. We address them in the following
pages. We enumerate their comments for ease of cross reference, include their comments
in italics and blue font, and provide our responses in plain text and black font.
We would like to thank the reviewers for their valuable und constructive comments.
Kind regards
Ingrid Felger
(on behalf of all co-authors)
*Reply to Reviewers Comments

2
Reviewer #1: The authors present the state of the art in malaria genotyping as it relates to
clinical trials for antimalarials. The message is an important one that has been overlooked by
clinical trialists, regulatory authorities and policy makers: current methods are limited and
may mis-attribute genotypes and inaccurately reflect drug treatments.
1.1 There are references here to unpublished results used as evidence and articles "in this
issue" which I was not able to discern (pages 6 para 2, references to Jones et al to which this
reviewer does not have a reference or access).
We have deleted this cross reference on page 8. It referred to a manuscript that was
submitted as a joint submission to Lancet Infectious Diseases together with this personal
views article. It was deemed unsuitable for LID and is now under review in Antimicrobial
Agents and Chemotherapy:
Jones, S., K. Kay, E.M. Hodel, S. Chy, A. Mbituymuremyi, A. Uwimana, D. Menard, I. Felger, I.
Hastings. Improving methods for analysing anti-malarial drug efficacy trials: molecular
correction based on length-polymorphic markers msp-1, msp-2 and glurp. Submitted.
The Jones et al. manuscript uses methodology previously published in:
Jaki T, Parry A, Winter K, Hastings I. Analysing malaria drug trials on a per-individual or per-
clone basis: a comparison of methods. Stat Med 2013; 32(17): 3020-38.
We therefore cite Jaki et al. 2013 (e.g. in caption to Figure 1) to allow readers access to the
underlying methodology. On page 8 of our article we have inserted (“Jones and Hastings,
personal communication”), which will be exchanged against the correct reference, should
the AAC article be published before this one.
Page 8: The paragraph on modelling has been revised as follows:
Modelling provides a new approach to validate potential algorithms for
interpreting PCR-corrected data. Pharmacological models can simulate a population
of patients in a drug efficacy trial, their therapeutic outcomes, and the genotyping
results that would occur at Day 0 and Day X.16 PCR-corrected failure rates were
compared using several molecular correction approaches based on simulation
models from Jaki and coworkers.16 The “2/3 approach” (i.e. only using glurp if msp1
and msp2 gave concordant results) provided the best fit with the simulated
treatment failure rates in a comparison to other molecular correction strategies
(Figure 1). Adopting the 2/3 approach instead of the current WHO sequential typing
method led to higher failure rate estimates but these were closer to the theoretical
true failure rate.
The current WHO/MMV method, and the new algorithms currently under
investigation (Jones and Hastings, personal communication), all seek to define
recurrent infections as either NI or a drug failure.

3
1.2 False impressions of drug failure in a clinical trial context is a major issue. Newer
methods including NGS are touched upon as possibly being the solution. The major concern I
have is that the authors would do well to convene a technical consultation with experts in
the field including clinicians, method developers, NGOs in trials like MMV, policy makers etc.
and then publish the results of the round table rather than this somewhat superficial piece.
The "2/3 approach" is mentioned as a solution but there is insufficient evidence that it
should be adopted without a fuller application to larger datasets beyond what is presented
in Figure 1.
We agree entirely with the comments listed by the Reviewer as “major concerns”. We
reassure the Reviewer as follows:
We wrote the article for LID primarily because we believe it will be of significant interest to
the readership of LID, as it deals with two crucial facets of malaria control, namely drug
efficacy trials and monitoring for emergent drug resistance. However, as recognised by the
Reviewer, it also has a significant political aspect. We agree with the Reviewer that a
“technical consultation” and “round table” discussion of key stakeholders will be necessary
to reach a consensus on most appropriate methodology and we plan to organise and
convene such a meeting. The problem is that such meetings require financial support and
clear demonstration to donors and potential participants that there is a clear need for such
a meeting; this article is designed to start this process. We are ideally placed to drive this
process. Current recommendations originate from round table technical discussions held in
Geneva in May 2007, supported by WHO and Medicines for Malaria Venture (MMV) and
subsequently published as a WHO report i.e.
World Health Organization (2008). Methods and techniques for clinical trials on antimalarial
drug efficacy: genotyping to identify parasite populations.
H-P Beck chaired that meeting, I. Felger was rapporteur, J. Möhrle was part of the
secretariat and G. Snounou was a participant. More recently I. Hastings was asked by WHO
via their TEG on Drug Resistance and Containment, to asses and evaluate the literature and
methodology of correction in trials. We anticipate that this submission to LID would be the
first stage of the process leading to a similar meeting being convened in the near future. We
recognise that support for policy change, driven by scientific analysis, is one of the prime
objectives of LID, hence our decision to submit in your journal.
In addition, I. Felger and I. Hastings participated in the Technical Expert Group on Drug
Efficacy and Response held at the World Health Organization, Geneva, Switzerland in June
2017. The group discussed the issue of molecular correction and their key recommendation
was: ““The Technical Expert Group (TEG) recommends that once the new analysis has been
completed, the guidance on P. falciparum genotyping should be reviewed and revised if
necessary” (Minutes available at https://www.who.int/malaria/mpac/mpac-oct2017-teg-drug-
efficacy-response-session3.pdf?ua=1.)

4
Page 10: As recommended by Reviewer 1 we suggest at the end of our paper that it is timely
to convene another technical meeting to update recommendations for PCR-correction
published in 2008 by WHO.
We suggest that improved understanding and technical developments since
publication of the WHO/MMV consensus protocols (2008) makes it important that
its recommendations should be updated. Another technical meeting should be
convened, particularly to address the methodological requirements for drug
registration trials that are more demanding than drug resistance monitoring studies.
Regulatory trials require precise methodologies and should be implemented as state-
of-the-art. The present paper will serve as a basis for discussion towards a revised
consensus.

Citations
More filters
Journal ArticleDOI
Christopher G Jacob1, Nguyen Thuy-Nhien2, Mayfong Mayxay3, Mayfong Mayxay4, Mayfong Mayxay2, Richard J. Maude5, Richard J. Maude2, Richard J. Maude6, Huynh Hong Quang, Bouasy Hongvanthong, Viengxay Vanisaveth, Thang Ngo Duc, Huy Rekol, Rob W. van der Pluijm6, Rob W. van der Pluijm2, Lorenz von Seidlein2, Lorenz von Seidlein6, Rick M. Fairhurst7, François Nosten2, Amir Hossain8, Naomi Park1, Scott Goodwin1, Pascal Ringwald9, Keobouphaphone Chindavongsa, Paul N. Newton3, Paul N. Newton2, Paul N. Newton6, Elizabeth A. Ashley2, Elizabeth A. Ashley3, Sonexay Phalivong3, Rapeephan R. Maude6, Rithea Leang, Cheah Huch, Le Thanh Dong, Kim-Tuyen Nguyen2, Tran Minh Nhat2, Tran Tinh Hien2, Hoa Nguyen, Nicole Zdrojewski, Sara E. Canavati, Abdullah Abu Sayeed8, Didar Uddin6, Caroline O. Buckee5, Caterina I. Fanello6, Caterina I. Fanello2, Marie A. Onyamboko10, Thomas J. Peto6, Thomas J. Peto2, Rupam Tripura2, Rupam Tripura6, Chanaki Amaratunga7, Aung Myint Thu2, Gilles Delmas2, Jordi Landier11, Daniel M. Parker12, Nguyen Hoang Chau2, Dysoley Lek, Seila Suon, James J Callery6, James J Callery2, Podjanee Jittamala6, Borimas Hanboonkunupakarn6, Sasithon Pukrittayakamee6, Sasithon Pukrittayakamee13, Aung Pyae Phyo2, Frank Smithuis2, Khin Lin, Myo Thant, Tin Maung Hlaing, Parthasarathi Satpathi14, Sanghamitra Satpathi, Prativa K Behera, Amar Tripura, Subrata Baidya, Neena Valecha15, Anupkumar R. Anvikar15, Akhter ul Islam, Abul Faiz, Chanon Kunasol6, Eleanor Drury1, Mihir Kekre1, Mozam Ali1, Katie Love1, Shavanthi Rajatileka1, Anna E. Jeffreys16, Kate Rowlands16, Christina Hubbart16, Mehul Dhorda6, Mehul Dhorda2, Ranitha Vongpromek6, Namfon Kotanan6, Phrutsamon Wongnak6, Jacob Almagro Garcia2, Richard D. Pearson1, Richard D. Pearson2, Cristina V. Ariani1, Thanat Chookajorn6, Cinzia Malangone1, Thuy Nguyen1, Jim Stalker1, Ben Jeffery2, Jonathan Keatley1, Kimberly J. Johnson2, Kimberly J. Johnson1, Dawn Muddyman1, Xin Hui S Chan2, Xin Hui S Chan6, John Sillitoe1, Roberto Amato1, Victoria Simpson1, Victoria Simpson2, Sónia Gonçalves1, Kirk A. Rockett16, Kirk A. Rockett1, Nicholas P. J. Day6, Nicholas P. J. Day2, Arjen M. Dondorp6, Arjen M. Dondorp2, Dominic P. Kwiatkowski2, Dominic P. Kwiatkowski1, Olivo Miotto1, Olivo Miotto2, Olivo Miotto6 
10 Aug 2021-eLife
TL;DR: GenRe-Mekong as mentioned in this paper is a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures.
Abstract: Author(s): Jacob, Christopher G; Thuy-Nhien, Nguyen; Mayxay, Mayfong; Maude, Richard J; Quang, Huynh Hong; Hongvanthong, Bouasy; Vanisaveth, Viengxay; Ngo Duc, Thang; Rekol, Huy; van der Pluijm, Rob; von Seidlein, Lorenz; Fairhurst, Rick; Nosten, Francois; Hossain, Md Amir; Park, Naomi; Goodwin, Scott; Ringwald, Pascal; Chindavongsa, Keobouphaphone; Newton, Paul; Ashley, Elizabeth; Phalivong, Sonexay; Maude, Rapeephan; Leang, Rithea; Huch, Cheah; Dong, Le Thanh; Nguyen, Kim-Tuyen; Nhat, Tran Minh; Hien, Tran Tinh; Nguyen, Hoa; Zdrojewski, Nicole; Canavati, Sara; Sayeed, Abdullah Abu; Uddin, Didar; Buckee, Caroline; Fanello, Caterina I; Onyamboko, Marie; Peto, Thomas; Tripura, Rupam; Amaratunga, Chanaki; Myint Thu, Aung; Delmas, Gilles; Landier, Jordi; Parker, Daniel M; Chau, Nguyen Hoang; Lek, Dysoley; Suon, Seila; Callery, James; Jittamala, Podjanee; Hanboonkunupakarn, Borimas; Pukrittayakamee, Sasithon; Phyo, Aung Pyae; Smithuis, Frank; Lin, Khin; Thant, Myo; Hlaing, Tin Maung; Satpathi, Parthasarathi; Satpathi, Sanghamitra; Behera, Prativa K; Tripura, Amar; Baidya, Subrata; Valecha, Neena; Anvikar, Anupkumar R; Ul Islam, Akhter; Faiz, Abul; Kunasol, Chanon; Drury, Eleanor; Kekre, Mihir; Ali, Mozam; Love, Katie; Rajatileka, Shavanthi; Jeffreys, Anna E; Rowlands, Kate; Hubbart, Christina S; Dhorda, Mehul; Vongpromek, Ranitha; Kotanan, Namfon; Wongnak, Phrutsamon; Almagro Garcia, Jacob; Pearson, Richard D; Ariani, Cristina V; Chookajorn, Thanat; Malangone, Cinzia; Nguyen, T; Stalker, Jim; Jeffery, Ben | Abstract: BackgroundNational Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures.MethodsSamples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs.ResultsGenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs.ConclusionsGenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community.FundingThe GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

41 citations

Journal ArticleDOI
TL;DR: Deep amplicon sequencing (AmpSeq) was employed to improve sensitivity and reliability of minority clone detection in antimalarial drug trials and was highly reproducible with consistent quantification of co-infecting parasite clones within a host.
Abstract: Clinical trials monitoring malaria drug resistance require genotyping of recurrent Plasmodium falciparum parasites to distinguish between treatment failure and new infection occurring during the trial follow up period. Because trial participants usually harbour multi-clonal P. falciparum infections, deep amplicon sequencing (AmpSeq) was employed to improve sensitivity and reliability of minority clone detection. Paired samples from 32 drug trial participants were Illumina deep-sequenced for five molecular markers. Reads were analysed by custom-made software HaplotypR and trial outcomes compared to results from the previous standard genotyping method based on length-polymorphic markers. Diversity of AmpSeq markers in pre-treatment samples was comparable or higher than length-polymorphic markers. AmpSeq was highly reproducible with consistent quantification of co-infecting parasite clones within a host. Outcomes of the three best-performing markers, cpmp, cpp and ama1-D3, agreed in 26/32 (81%) of patients. Discordance between the three markers performed per sample was much lower by AmpSeq (six patients) compared to length-polymorphic markers (eleven patients). Using AmpSeq for discrimination of recrudescence and new infection in antimalarial drug trials provides highly reproducible and robust characterization of clone dynamics during trial follow-up. AmpSeq overcomes limitations inherent to length-polymorphic markers. Regulatory clinical trials of antimalarial drugs will greatly benefit from this unbiased typing method.

33 citations

Posted ContentDOI
Christopher G Jacob1, Nguyen Thuy-Nhien2, Mayfong Mayxay3, Mayfong Mayxay2, Mayfong Mayxay4, Richard J. Maude5, Richard J. Maude6, Richard J. Maude2, Huynh Hong Quang, Bouasy Hongvanthong, Viengxay Vanisaveth, Thang Ngo Duc, Huy Rekol, Rob W. van der Pluijm5, Rob W. van der Pluijm2, Lorenz von Seidlein2, Lorenz von Seidlein5, Rick M. Fairhurst7, François Nosten2, Amir Hossain8, Naomi Park1, Scott Goodwin1, Pascal Ringwald9, Keobouphaphone Chindavongsa, Paul N. Newton3, Paul N. Newton5, Paul N. Newton2, Elizabeth A. Ashley3, Elizabeth A. Ashley2, Sonexay Phalivong3, Rapeephan R. Maude5, Rithea Leang, Cheah Huch, Le Thanh Dong, Kim-Tuyen Nguyen2, Tran Minh Nhat2, Tran Tinh Hien2, Hoa Nguyen, Nicole Zdrojewski, Sara E. Canavati, Abdullah Abu Sayeed8, Didar Uddin5, Caroline O. Buckee6, Caterina I. Fanello5, Caterina I. Fanello2, Marie A. Onyamboko10, Thomas J. Peto2, Thomas J. Peto5, Rupam Tripura2, Rupam Tripura5, Chanaki Amaratunga7, Aung Myint Thu2, Gilles Delmas2, Jordi Landier11, Nguyen Hoang Chau2, Dysoley Lek, Seila Suon, James J Callery5, James J Callery2, Podjanee Jittamala5, Borimas Hanboonkunupakarn5, Sasithon Pukrittayakamee12, Sasithon Pukrittayakamee5, Aung Pyae Phyo2, Frank Smithuis2, Khin Lin, Myo Thant, Tin Maung Hlaing, Parthasarathi Satpathi13, Sanghamitra Satpathi, Prativa K Behera, Amar Tripura, Subrata Baidya, Neena Valecha14, Anupkumar R. Anvikar14, Akhter ul Islam, Abul Faiz, Chanon Kunasol5, Eleanor Drury1, Mihir Kekre1, Mozam Ali1, Katie Love1, Shavanthi Rajatileka1, Anna E. Jeffreys15, Kate Rowlands15, Christina Hubbart15, Mehul Dhorda2, Mehul Dhorda5, Ranitha Vongpromek5, Namfon Kotanan5, Phrutsamon Wongnak5, Jacob Almagro Garcia2, Richard D. Pearson1, Richard D. Pearson2, Cristina V. Ariani1, Thanat Chookajorn5, Cinzia Malangone1, Thuy Nguyen1, Jim Stalker1, Ben Jeffery2, Jonathan Keatley1, Kimberly J. Johnson2, Kimberly J. Johnson1, Dawn Muddyman1, Xin Hui S Chan5, Xin Hui S Chan2, John Sillitoe1, Roberto Amato1, Victoria Simpson2, Victoria Simpson1, Sónia Gonçalves1, Kirk A. Rockett15, Kirk A. Rockett1, Nicholas P. J. Day5, Nicholas P. J. Day2, Arjen M. Dondorp2, Arjen M. Dondorp5, Dominic P. Kwiatkowski2, Dominic P. Kwiatkowski1, Olivo Miotto2, Olivo Miotto1, Olivo Miotto5 
25 Jul 2020-medRxiv
TL;DR: GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion, enables NMCPs to conduct large-scale surveillance project in endemic regions by integrating simple sample collection procedures in the routine operations of public health facilities.
Abstract: The use of parasite genetic data by National Malaria Control Programmes (NMCPs) is currently limited, and typically focused on specific genetic features or a small number of study sites. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS). By integrating simple sample collection procedures in the routine operations of public health facilities, GenRe-Mekong enables NMCPs to conduct large-scale surveillance project in endemic regions. Samples are processed by the SpotMalaria platform, which uses high-throughput technologies to produce a broad set of genotypes, including most known drug resistance markers, species markers and a genomic barcode. Through the application of heuristics based on published evidence, GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions that are used to map prevalence of resistance to multiple drugs. To date, GenRe-Mekong has worked with NMCPs in five countries, and with several large-scale research projects, processing 9,645 samples from clinical cases. The monitoring of resistance markers has been especially valuable for NMCPs tracking the recent rapid spread of DHA-piperaquine resistant parasites across the region. In Vietnam and Laos, data from GenRe-Mekong have provided novel knowledge about the spread of these resistant strains in provinces previously thought to be unaffected. GenRe-Mekong facilitates data sharing by aggregating at regional level results from different countries, providing cross-border views of the spread of resistant strains.

31 citations

Journal ArticleDOI
TL;DR: Differences in the duration of protection between the artemisinin combination therapies could alter population-level clinical incidence of malaria by up to 14% in under-5-year-old children when the drugs were used as first-line treatments in areas with high, seasonal transmission.
Abstract: The majority of Plasmodium falciparum malaria cases in Africa are treated with the artemisinin combination therapies artemether-lumefantrine (AL) and artesunate-amodiaquine (AS-AQ), with amodiaquine being also widely used as part of seasonal malaria chemoprevention programs combined with sulfadoxine-pyrimethamine. While artemisinin derivatives have a short half-life, lumefantrine and amodiaquine may give rise to differing durations of post-treatment prophylaxis, an important additional benefit to patients in higher transmission areas. We analyzed individual patient data from 8 clinical trials of AL versus AS-AQ in 12 sites in Africa (n = 4214 individuals). The time to PCR-confirmed reinfection after treatment was used to estimate the duration of post-treatment protection, accounting for variation in transmission intensity between settings using hidden semi-Markov models. Accelerated failure-time models were used to identify potential effects of covariates on the time to reinfection. The estimated duration of chemoprophylaxis was then used in a mathematical model of malaria transmission to determine the potential public health impact of each drug when used for first-line treatment. We estimated a mean duration of post-treatment protection of 13.0 days (95% CI 10.7–15.7) for AL and 15.2 days (95% CI 12.8–18.4) for AS-AQ overall. However, the duration varied significantly between trial sites, from 8.7–18.6 days for AL and 10.2–18.7 days for AS-AQ. Significant predictors of time to reinfection in multivariable models were transmission intensity, age, drug, and parasite genotype. Where wild type pfmdr1 and pfcrt parasite genotypes predominated ( 80%), AL provided up to 1.5-fold longer protection than AS-AQ. Our simulations found that these differences in the duration of protection could alter population-level clinical incidence of malaria by up to 14% in under-5-year-old children when the drugs were used as first-line treatments in areas with high, seasonal transmission. Choosing a first-line treatment which provides optimal post-treatment prophylaxis given the local prevalence of resistance-associated markers could make a significant contribution to reducing malaria morbidity.

27 citations

Journal ArticleDOI
TL;DR: Results demonstrate that in vivo drug activity of artesunate is faster than is indicated by the PC half-life, which is currently used to assess antimalarial activity of novel agents and to monitor resistance.
Abstract: BACKGROUND Artemisinin derivatives are the leading class of antimalarial drugs due to their rapid onset of action and rapid clearance of circulating parasites. The parasite clearance half-life measures the rate of loss of parasites from blood after treatment, and this is currently used to assess antimalarial activity of novel agents and to monitor resistance. However, a number of recent studies have challenged the use of parasite clearance to measure drug activity, arguing that many circulating parasites may be nonviable. METHODS Plasmodium falciparum-infected subjects (n = 10) in a malaria volunteer infection study were administered a single dose of artesunate (2 mg/kg). Circulating parasite concentration was assessed by means of quantitative polymerase chain reaction (qPCR). Parasite viability after artesunate administration was estimated by mathematical modeling of the ex vivo growth of parasites collected from subjects. RESULTS We showed that in artemisinin-sensitive infection, viable parasites declined to <0.1% of baseline within 8 hours after artesunate administration, while the total number of circulating parasites measured with quantitative polymerase chain reaction remained unchanged. In artemisinin-resistant infections over the same interval, viable parasites declined to 51.4% (standard error of the mean, 4.6%) of baseline. CONCLUSIONS These results demonstrate that in vivo drug activity of artesunate is faster than is indicated by the parasite clearance half-life.

9 citations

References
More filters
Journal ArticleDOI
TL;DR: How control measures that aim to reduce malaria transmission, such as mass drug administration and a transmission-blocking vaccine, might better be deployed are drawn together to show how the application of molecular techniques has led to the identification of submicroscopic gametocyte carriage and to a reassessment of the human infectious reservoir.
Abstract: Malaria remains a major cause of morbidity and mortality in the tropics, with Plasmodium falciparum responsible for the majority of the disease burden and P. vivax being the geographically most widely distributed cause of malaria. Gametocytes are the sexual-stage parasites that infect Anopheles mosquitoes and mediate the onward transmission of the disease. Gametocytes are poorly studied despite this crucial role, but with a recent resurgence of interest in malaria elimination, the study of gametocytes is in vogue. This review highlights the current state of knowledge with regard to the development and longevity of P. falciparum and P. vivax gametocytes in the human host and the factors influencing their distribution within endemic populations. The evidence for immune responses, antimalarial drugs, and drug resistance influencing infectiousness to mosquitoes is reviewed. We discuss how the application of molecular techniques has led to the identification of submicroscopic gametocyte carriage and to a reassessment of the human infectious reservoir. These components are drawn together to show how control measures that aim to reduce malaria transmission, such as mass drug administration and a transmission-blocking vaccine, might better be deployed.

651 citations

Journal ArticleDOI
TL;DR: Protocols for characterization of 12 microsatellite markers from finger-prick blood samples infected with Plasmodium falciparum are described, which allow amplification from low levels of template, while eliminating the problem of spurious products due to primer carry over from the primary round of PCR.
Abstract: Multiple, selectively neutral genetic markers are the most appropriate tools for analysis of parasite population structure and epidemiology, but yet existing methods for characterization of malaria field samples utilize a limited number of antigen encoding genes, which appear to be under strong selection. We describe protocols for characterization of 12 microsatellite markers from finger-prick blood samples infected with Plasmodium falciparum. A two-step, heminested strategy was used to amplify all loci, and products were visualized by fluorescent end-labelling of internal primers. This procedure allows amplification from low levels of template, while eliminating the problem of spurious products due to primer carry over from the primary round of PCR. The loci can be conveniently multiplexed, while accurate sizing and quantification of PCR products can be automated using the GENOTYPER software. The primers do not amplify co-infecting malaria species such as P. vivax and P. malariae. To demonstrate the utility of these markers, we characterized 57 infected finger-prick blood samples from the village of Mebat in Papua New Guinea for all 12 loci, and all samples were genotyped a second time to measure reproducibility. Numbers of alleles per locus range from 4 to 10 in this population, while heterozygosities range from 0.21 to 0.87. Reproducibility (measured as concordance between predominant alleles detected in replicate samples) ranged from 92 to 98% for the 12 loci. The composition of PCR products from infections containing multiple malaria clones could also be defined using strict criteria and scored in a highly repeatable manner.

332 citations

Journal ArticleDOI
TL;DR: In the eight children harboring P. falciparum throughout the study period, infections were found to be highly complex with daily changes in both parasite density and genotypic pattern, and 48-hr periodicity in these fluctuations suggests that P. Falconerum infections consist of inherently synchronous subpopulations of parasites.
Abstract: Plasmodium falciparum is the major cause of malaria morbidity and mortality in the world. Biologic and antigenic diversity is a characteristic of this parasite and infections can consist of several genetically diverse parasites. The daily dynamics of these parasite subpopulations were investigated in asymptomatic children in rural Tanzania. Fingerprick blood samples were collected on 14 consecutive days from 20 children. Parasite densities were detected by light microscopy and genotyping of P. falciparum was done using a nested polymerase chain reaction (PCR) assay targeting polymorphic regions on the merozoite surface protein-1 (MSP-1), MSP-2, and glutamine-rich protein (GLURP) genes. In the eight children harboring P. falciparum throughout the study period, infections were found to be highly complex with daily changes in both parasite density and genotypic pattern. A nonrandom, 48-hr periodicity in these fluctuations suggests that P. falciparum infections consist of inherently synchronous subpopulations of parasites. These findings have important biologic and epidemiologic implications since one blood sample may only partly reflect the whole parasite population in an infected individual.

239 citations

Journal ArticleDOI
TL;DR: It is suggested that although genotyping can provide strong evidence for differentiating between true recrudescence and reinfection, it must be interpreted with caution and proposed strategies that might help minimize these uncertainties.

205 citations

Journal ArticleDOI
TL;DR: Results show that immunization with SPf66 modulates the course of naturally occurring infections, as reflected by reduced MOIs, and provide further evidence that premunition from concurrent infections is important in immunity against clinical malaria.
Abstract: In the first phase III efficacy trial of the malaria vaccine SPf66 in Africa, MOIs in SPf66- and placebo-vaccinated children were analyzed by polymerase chain reaction-restriction fragment length polymorphism of the Plasmodium falciparum merozoite surface antigen 2 (MSA2). MOIs were significantly reduced in asymptomatic vaccine recipients compared with those in asymptomatic placebo recipients; however, no differences were observed among symptomatic children in the vaccine and control groups. These results show that immunization with SPf66 modulates the course of naturally occurring infections, as reflected by reduced MOIs. In placebo recipients, however, there was a significant negative correlation between numbers of infecting genotypes, as identified by MSA2, and morbidity. Asymptomatic placebo recipients had an average of 5 concurrent infections, whereas children with clinical cases had an average of 3.4 infections. These data provide further evidence that premunition from concurrent infections is important in immunity against clinical malaria. No such effect of multiple infections was found in the vaccinated group.

192 citations

Frequently Asked Questions (2)
Q1. What are the contributions in this paper?

The authors describe and discuss recent developments in these genotyping strategies with particular focus on method validation and limitations. The authors aim to promote discussion around these issues and argue for the adoption of improved standardized PCR-correction methodologies. 

For critical discussion and sharing their knowledge the authors wish to thank other experts in the field who supported their work. For critical discussion and sharing their knowledge the authors wish to thank other experts in the field who supported their work. If both markers give congruent results ( either 10 Recrudescence or NI ), no further marker needs be typed ; in the case of discrepant results, one additional marker should be added ( either glurp or new marker ) ; if the “ 2/3 approach ” is not possible one should classify the episode as a recrudescence, a stringent interpretation that avoids underestimation of treatment failures. The authors suggest that improved understanding and technical developments since publication of the WHO/MMV consensus protocols ( 2008 ) makes it important that its recommendations should be updated. 

Trending Questions (1)
What method/ strategies did they do to go back to normal when people were infected with malaria?

The authors discuss the use of PCR correction strategies, specifically comparing parasite genotypes before and after treatment, to accurately determine drug failure rates in malaria drug trials.