Date Published: January 11, 2016
Publisher: Public Library of Science
Author(s): M. Andreína Pacheco, Mary Lopez-Perez, Andrés F. Vallejo, Sócrates Herrera, Myriam Arévalo-Herrera, Ananias A. Escalante, Photini Sinnis. http://doi.org/10.1371/journal.pntd.0004355
Abstract: BackgroundMultiplicity of infection (MOI) refers to the average number of distinct parasite genotypes concurrently infecting a patient. Although several studies have reported on MOI and the frequency of multiclonal infections in Plasmodium falciparum, there is limited data on Plasmodium vivax. Here, MOI and the frequency of multiclonal infections were studied in areas from South America where P. vivax and P. falciparum can be compared.Methodology/Principal FindingsAs part of a passive surveillance study, 1,328 positive malaria patients were recruited between 2011 and 2013 in low transmission areas from Colombia. Of those, there were only 38 P. vivax and 24 P. falciparum clinically complicated cases scattered throughout the time of the study. Samples from uncomplicated cases were matched in time and location with the complicated cases in order to compare the circulating genotypes for these two categories. A total of 92 P. vivax and 57 P. falciparum uncomplicated cases were randomly subsampled. All samples were genotyped by using neutral microsatellites. Plasmodium vivax showed more multiclonal infections (47.7%) than P. falciparum (14.8%). Population genetics and haplotype network analyses did not detect differences in the circulating genotypes between complicated and uncomplicated cases in each parasite. However, a Fisher exact test yielded a significant association between having multiclonal P. vivax infections and complicated malaria. No association was found for P. falciparum infections.ConclusionThe association between multiclonal infections and disease severity in P. vivax is consistent with previous observations made in rodent malaria. The contrasting pattern between P. vivax and P. falciparum could be explained, at least in part, by the fact that P. vivax infections have lineages that were more distantly related among them than in the case of the P. falciparum multiclonal infections. Future research should address the possible role that acquired immunity and exposure may have on multiclonal infections and their association with disease severity.
Partial Text: A common observation in many malaria endemic areas is that there are patients concurrently infected by more than one distinct parasite genotype. These infections are usually referred to as multiclonal infections. In addition to the frequency of multiclonal infections, molecular epidemiologists estimate the average number of lineages per infected individual or multiplicity of infection (MOI). Together, MOI and the frequency of multiclonal infections are measurements that relate to transmission intensity [1, 2]. Ecologically, multiclonal infections could be the result of two different processes, the co-transmission of different parasite variants (co-infections) or the overlap of genetic variants due to infectious contacts before the primary infection is resolved (superinfections) [1, 2]. Distinguishing between these processes is particularly laborious in field settings.
There were few cases of complicated malaria in these areas with low transmission . A total of 38 P. vivax and 24 P. falciparum cases were classified as clinically complicated following the criteria listed in Table 1. Uncomplicated cases were sub-sampled to create a control group that matched the complicate malaria cases (CMC) in terms of location and the time when the case was diagnosed. The age, gender, ethnic composition, average MOI (and range) and the percentage of multiplicity of infection of the complicated and uncomplicated malaria cases are reported in Table 2. No noticeable demographic differences were observed between the complicated and uncomplicated malaria groups (Table 2) and no association between gender and complicated and uncomplicated malaria cases was found (Table 3).
There have been multiple epidemiological investigations aiming to explore the relationship between MOI and/or the frequency of multiclonal infections with variables of epidemiological interest, including but not limited to clinical endpoints. Examples of such studies are shown in S1 Table. The variation of the genetic markers used and the broad spectrum of epidemiological variables investigated hampered our ability to compare findings across studies. Nevertheless, there were some emerging patterns. For example, in the handful of studies where P. vivax was compared with P. falciparum, patients with P. vivax malaria harbored multiclonal infections more often than those with P. falciparum malaria [8, 11, 13, 45, 46](S1 Table). Our findings are consistent with this global trend. The observed higher frequency of multiclonal infections in P. vivax could be the result of hypnozoites accumulating in the liver yielding multiple relapses of distinct genotypes. If this were the only factor, it would imply that patients received incomplete treatment with primaquine, a drug that is prescribed to treat uncomplicated P. vivax malaria in Colombia and other Latin-American countries. Our observation, however, cannot be taken as evidence of lack of compliance with the local drug policy. It is possible that P. vivax patients remained asymptomatic for a long period of time  facilitating superinfections because antimalarial treatment was not provided. A factor that could also contribute to this pattern is that P. vivax has higher prevalence and genetic diversity in this region when compared to P. falciparum; thus, ecological differences in terms of transmission are easier to detect and could partially explain the higher frequency of multiclonal infections as a result of coinfections or superinfections [29, 30]. The differential contribution of these and other factors to the observed high frequency of multiclonal infections in P. vivax is a matter that requires additional investigations.