Abstract
BackgroundParasite genetic diversity and multiplicity of infection (MOI) affect clinical outcomes, response to drug treatment and naturally-acquired or vaccine-induced immunity. Traditional methods often underestimate the frequency and diversity of multiclonal infections due to technical sensitivity and specificity. Next-generation sequencing techniques provide a novel opportunity to study complexity of parasite populations and molecular epidemiology.MethodsSymptomatic and asymptomatic Plasmodium vivax samples were collected from health centres/hospitals and schools, respectively, from 2011 to 2015 in Ethiopia. Similarly, both symptomatic and asymptomatic Plasmodium falciparum samples were collected, respectively, from hospitals and schools in 2005 and 2015 in Kenya. Finger-pricked blood samples were collected and dried on filter paper. Long amplicon (> 400 bp) deep sequencing of merozoite surface protein 1 (msp1) gene was conducted to determine multiplicity and molecular epidemiology of P. vivax and P. falciparum infections. The results were compared with those based on short amplicon (117 bp) deep sequencing.ResultsA total of 139 P. vivax and 222 P. falciparum samples were pyro-sequenced for pvmsp1 and pfmsp1, yielding a total of 21 P. vivax and 99 P. falciparum predominant haplotypes. The average MOI for P. vivax and P. falciparum were 2.16 and 2.68, respectively, which were significantly higher than that of microsatellite markers and short amplicon (117 bp) deep sequencing. Multiclonal infections were detected in 62.2% of the samples for P. vivax and 74.8% of the samples for P. falciparum. Four out of the five subjects with recurrent P. vivax malaria were found to be a relapse 44–65 days after clearance of parasites. No difference was observed in MOI among P. vivax patients of different symptoms, ages and genders. Similar patterns were also observed in P. falciparum except for one study site in Kenyan lowland areas with significantly higher MOI.ConclusionsThe study used a novel method to evaluate Plasmodium MOI and molecular epidemiological patterns by long amplicon ultra-deep sequencing. The complexity of infections were similar among age groups, symptoms, genders, transmission settings (spatial heterogeneity), as well as over years (pre- vs. post-scale-up interventions). This study demonstrated that long amplicon deep sequencing is a useful tool to investigate multiplicity and molecular epidemiology of Plasmodium parasite infections.
Highlights
Parasite genetic diversity and multiplicity of infection (MOI) affect clinical outcomes, response to drug treatment and naturally-acquired or vaccine-induced immunity
Relapses play an important role in the transmission of P. vivax in malaria endemic areas [4].With the scaling up of interventions since 2006, primarily mass distribution of insecticidetreated nets (ITNs), indoor residual spraying (IRS), and artemisinin-based combination therapy (ACT), malaria transmission has declined tremendously in the past decade [5]
Sequence reads and haplotype determination A total of 384 PCR reactions (362 samples and 2 controls as well as 20 replicate PCR reactions) were successfully amplified and sequenced, resulting in 166 M total reads, of which 120 M reads (74%) passed filter, including 100 M with Qscores > 30, with an exception of three P. vivax samples and two P. falciparum samples with less than 1000 reads that were excluded from the analyses
Summary
Parasite genetic diversity and multiplicity of infection (MOI) affect clinical outcomes, response to drug treatment and naturally-acquired or vaccine-induced immunity. In 2016, there were an estimated 216 million cases and 445,000 deaths of malaria occurred worldwide; and nearly half of the world’s population lived in 91 countries and territories are at risk of malaria transmission [1]. The majority of malaria cases and deaths (~ 90%) occur in sub-Saharan Africa. Plasmodium falciparum is the most prevalent malaria parasite in sub-Saharan Africa, while Plasmodium vivax is the most widespread human malaria with approximately 2.5 billion people at risk of infection worldwide [2]. Relapses play an important role in the transmission of P. vivax in malaria endemic areas [4].With the scaling up of interventions since 2006, primarily mass distribution of insecticidetreated nets (ITNs), indoor residual spraying (IRS), and artemisinin-based combination therapy (ACT), malaria transmission has declined tremendously in the past decade [5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
