[Show abstract][Hide abstract] ABSTRACT: Plasmodium vivax is the geographically most widespread human malaria parasite. To analyze patterns of microsatellite diversity and population structure across countries of different transmission intensity, genotyping data from 11 microsatellite markers was either generated or compiled from 841 isolates from four continents collected in 1999-2008. Diversity was highest in South-East Asia (mean allelic richness 10.0-12.8), intermediate in the South Pacific (8.1-9.9) Madagascar and Sudan (7.9-8.4), and lowest in South America and Central Asia (5.5-7.2). A reduced panel of only 3 markers was sufficient to identify approx. 90% of all haplotypes in South Pacific, African and SE-Asian populations, but only 60-80% in Latin American populations, suggesting that typing of 2-6 markers, depending on the level of endemicity, is sufficient for epidemiological studies. Clustering analysis showed distinct clusters in Peru and Brazil, but little sub-structuring was observed within Africa, SE-Asia or the South Pacific. Isolates from Uzbekistan were exceptional, as a near-clonal parasite population was observed that was clearly separated from all other populations (FST>0.2). Outside Central Asia FST values were highest (0.11-0.16) between South American and all other populations, and lowest (0.04-0.07) between populations from South-East Asia and the South Pacific. These comparisons between P. vivax populations from four continents indicated that not only transmission intensity, but also geographical isolation affect diversity and population structure. However, the high effective population size results in slow changes of these parameters. This persistency must be taken into account when assessing the impact of control programs on the genetic structure of parasite populations.
[Show abstract][Hide abstract] ABSTRACT: In Vietnam the malaria burden has been drastically reduced over the past two decades but Plasmodium vivax is becoming increasingly important mainly due to its relapsing nature and the difficulty to radically cure dormant parasites from the liver. A two-year cohort study was conducted in Central Vietnam to assess the efficacy of the radical cure regimen base on a 10-day Primaquine (0.5mg/kg/d) in combination with the standard 3-day chloroquine (total 25mg/kg) regimen. We report the genetic diversity and population structure of P. vivax infections before and after radical treatment. All day 0 (n=247) and post treatment P. vivax infections (n=788) detected by microscopy and PCR during the 2-year monthly follow-up were genotyped using 16 previously described microsatellites. Genetic diversity, linkage disequilibrium, population structure and haplotype clustering were analyzed in post treatment samples and compared to Day 0 samples. All markers were highly polymorphic with 3 to 30 alleles per marker and heterozigosity (He) values ranging from 0.35 to 0.90. Overall He values were not significantly different between day 0 and posttreatment samples (He = 0.64 and 0.66 respectively). In addition, 71.0% of all infections were polyclonal (76.9% at day 0 vs. 69.2% post-treatment samples) and the average multiplicity of infection (MOI) was 1.9 parasites/ person (MOI = 2.1 at D0, MOI =1.8 at recurrences). Genetic diversity of parasite population experimented significant changes when parasite population before treatment was compared with parasite population in the second year follow up (FST= 0.21). which may suggest a delayed effect of the intervention or may reflect the intense follow up (with treatment of all cases) study design. In order to estimate multilocus linkage disequilibrium (LD) changes between day 0 and post-treatment samples, we calculated the index of association IsA (which is zero for LD). We observed higher LD in post-treatment than day 0 parasite population (IsA = 0.093, P=0.0001 and IsA = 0.039, P=0.0001, respectively), suggesting inbreeding and a clonal population structure. Overall parasite population in the study is genetically diverse, and has a low effective recombination rate that contrasts with the high number of polyclonal infections.
ASTMH 63rd Annual Meeting, New Orleans, LA, USA; 11/2014
[Show abstract][Hide abstract] ABSTRACT: Despite the large burden of Plasmodium vivax, little is known about its transmission dynamics. This study explored the population structure and spatio-temporal dynamics of P. vivax recurrent infections after radical cure in a two-year cohort study carried out in a rural community of the Peruvian Amazon.
A total of 37 P. vivax participants recruited in San Carlos community (Peru) between April and December 2008 were treated radically with chloroquine and primaquine and followed up monthly for two years with systematic blood sampling. All samples were screened for malaria parasites and subsequently all P. vivax infections genotyped using 15 microsatellites. Parasite population structure and dynamics were determined by computing different genetic indices and using spatio-temporal statistics.
After radical cure, 76% of the study participants experienced one or more recurrent P. vivax infections, most of them sub-patent and asymptomatic. The parasite population displayed limited genetic diversity (He = 0.49) and clonal structure, with most infections (84%) being monoclonal. Spatio-temporal clusters of specific haplotypes were found throughout the study and persistence of highly frequent haplotypes were observed over several months within the same participants/households.
In San Carlos community, P. vivax recurrences were commonly observed after radical treatment, and characterized by asymptomatic, sub-patent and clustered infections (within and between individuals from a few neighbouring households). Moreover low genetic diversity as well as parasite inbreeding are likely to define a clonal parasite population which has important implications on the malaria epidemiology of the study area.
[Show abstract][Hide abstract] ABSTRACT: Introduction Despite the large burden of Plasmodium vivax, little is known about its transmission dynamics. The study of the parasite population structure and dynamics give insights on particular epidemiological features of malaria in endemic areas. We explored the population structure and spatio-temporal dynamics of P. vivax infections in a 2-year cohort study carried out in a rural community of the Peruvian Amazon.
Materials and Methods Between March and December 2008, 37 P. vivax patients were treated with chloroquine and primaquine and followed up monthly for 2 years with systematic blood sampling. All samples were screened by microscopy and species-specific PCR for malaria parasites and subsequently all P. vivax infections genotyped using 15 microsatellites. Parasitepopulation structure and dynamics were determined by computing different genetic indices and using spatio-temporal statistics.
Results Seventy-six percent of the study participants experienced one or more recurrent P. vivax infections during their follow-up. Fifty-five percent of the recurrences were subpatent and asymptomatic. The P. vivax population displayed limited overall genetic diversity (He = 0.49), high frequency of monoclonal infections (84%), presence of linkage disequilibrium (LD) and low probability of outbreeding (Psex<0.0001). Up to 20 unique haplotypes unequally distributed in four haplogroups were found. Haplotype replacement was reflected by spatiotemporal clustering and changes on the degree of LD and the genetic diversity after the first year of follow up (P < 0.001).
Conclusions The parasite population and dynamics found in this study may explain some epidemiologic features of the study area: (i) the clonal parasite population and haplotype clustering could be favored by the geographical isolation of the study area; (ii) continuous infections with the same clones may facilitate the development of clinical immunity, reflected on high recurrence of asymptomatic infections; (iii) the high rate of recurrent infections after treatment could be associated to the increased risk of spread of drug resistance traits in clonal populations.
8th European Congress on Tropical Medicine and International Health, Copenhagen; 09/2013
[Show abstract][Hide abstract] ABSTRACT: We have modified an existing semi-nested multiplex polymerase chain reaction (PCR) by adding one Plasmodium knowlesi-specific nested PCR, and validated the latter against laboratory and clinical samples. This new method has the advantage of being relatively affordable in low resource settings while identifying the five human Plasmodium species with a three-step PCR.
The American journal of tropical medicine and hygiene 08/2013; 89(4). DOI:10.4269/ajtmh.13-0027 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Vietnam, Plasmodium falciparum and P. vivax are responsible for most malaria infections, and P. malariae and P. ovale infections are rarely reported. Nevertheless, species-specific polymerase chain reaction analysis on 2,303 blood samples collected during a cross-sectional survey conducted in a forest area of central Vietnam identified 223 (9.7%) P. falciparum, 170 (7.4%) P. vivax, 95 (4.1%) P. malariae, and 19 (0.8%) P. ovale mono-infections and 164 (7.1%) mixed infections. Of the 671 Plasmodium-positive samples by polymerase chain reaction, only 331 were detected by microscopy. Microscopy poorly diagnosed P. malariae, P. ovale, and mixed infections. Clinical and sub-clinical infections occurred in all age groups. The risk for infection and disease decreased with age, probably because of acquired partial immunity. The common occurrence of sub-patent infections seems to indicate that the malaria burden is underestimated and that diagnostic and therapeutic policies should be adapted accordingly.
The American journal of tropical medicine and hygiene 11/2012; 87(6). DOI:10.4269/ajtmh.2012.12-0047 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of a system for the continuous culture of Plasmodium vivax in vitro would benefit from the use of reticulocytes derived from differentiated hematopoietic stem cells (HCS). At present, the need to use both fresh reticulocytes and fresh P. vivax isolates represents a major obstacle towards this goal, particularly for laboratories located in non-endemic countries. Here, we describe a new method for the cryopreservation of HSC-derived reticulocytes to be used for both P. falciparum and P. vivax invasion tests. Cryopreserved P. falciparum and P. vivax isolates could invade both fresh and cryopreserved HSC-derived reticulocytes with similar efficiency. This new technique allows the storage of HSC-derived reticulocytes which can be used for later invasion tests and represents an important step towards the establishment of a continuous P. vivax culture.
PLoS ONE 07/2012; 7(7):e40798. DOI:10.1371/journal.pone.0040798 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To present a new approach for estimating the "true prevalence" of malaria and apply it to datasets from Peru, Vietnam, and Cambodia.
Bayesian models were developed for estimating both the malaria prevalence using different diagnostic tests (microscopy, PCR & ELISA), without the need of a gold standard, and the tests' characteristics. Several sources of information, i.e. data, expert opinions and other sources of knowledge can be integrated into the model. This approach resulting in an optimal and harmonized estimate of malaria infection prevalence, with no conflict between the different sources of information, was tested on data from Peru, Vietnam and Cambodia.
Malaria sero-prevalence was relatively low in all sites, with ELISA showing the highest estimates. The sensitivity of microscopy and ELISA were statistically lower in Vietnam than in the other sites. Similarly, the specificities of microscopy, ELISA and PCR were significantly lower in Vietnam than in the other sites. In Vietnam and Peru, microscopy was closer to the "true" estimate than the other 2 tests while as expected ELISA, with its lower specificity, usually overestimated the prevalence.
Bayesian methods are useful for analyzing prevalence results when no gold standard diagnostic test is available. Though some results are expected, e.g. PCR more sensitive than microscopy, a standardized and context-independent quantification of the diagnostic tests' characteristics (sensitivity and specificity) and the underlying malaria prevalence may be useful for comparing different sites. Indeed, the use of a single diagnostic technique could strongly bias the prevalence estimation. This limitation can be circumvented by using a Bayesian framework taking into account the imperfect characteristics of the currently available diagnostic tests. As discussed in the paper, this approach may further support global malaria burden estimation initiatives.
PLoS ONE 02/2011; 6(2):e16705. DOI:10.1371/journal.pone.0016705 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is an increasing body of literature reporting treatment failure of the currently recommended radical treatment of Plasmodium vivax infections. As P. vivax is the main malaria species outside the African continent, emerging tolerance to its radical treatment regime could have major consequences in countries like Peru, where 80% of malaria cases are due to P. vivax. Here we describe the results of a 1-year longitudinal follow up of 51 confirmed P. vivax patients living around Iquitos, Peruvian Amazon, and treated according to the Peruvian national guidelines.
Each month a blood sample for microscopy and later genotyping was systematically collected. Recent exposure to infection was estimated by detecting antibodies against the P. vivax circumsporozoite protein (CSP) and all PCR confirmed P. vivax infections were genotyped with 16 polymorphic microsatellites.
During a 1-year period, 84 recurrent infections, 22 positive also by microscopy, were identified, with a median survival time to first recurrent infection of 203 days. Most of them (71%) were asymptomatic; in 13 patients the infection persisted undetected by microscopy for several consecutive months. The genotype of mostly recurrent infections differed from that at day 0 while fewer differences were seen between the recurrent infections. The average expected heterozygosity was 0.56. There was strong linkage disequilibrium (I(A)(s) = 0.29, p<1.10(-4)) that remained also when analyzing only the unique haplotypes, suggesting common inbreeding.
In Peru, the P. vivax recurrent infections were common and displayed a high turnover of parasite genotypes compared to day 0. Plasmodium vivax patients, even when treated according to the national guidelines, may still represent an important parasite reservoir that can maintain transmission. Any elimination effort should consider such a hidden reservoir.
PLoS ONE 01/2011; 6(1):e16257. DOI:10.1371/journal.pone.0016257 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peru is one of the Latin American countries with the highest malaria burden, mainly due to Plasmodium vivax infections. However, little is known about P. vivax transmission dynamics in the Peruvian Amazon, where most malaria cases occur. The genetic diversity and population structure of P. vivax isolates collected in different communities around Iquitos city, the capital of the Peruvian Amazon, was determined.
Plasmodium vivax population structure was determined by multilocus genotyping with 16 microsatellites on 159 P. vivax infected blood samples (mono-infections) collected in four sites around Iquitos city. The population characteristics were assessed only in samples with monoclonal infections (n = 94), and the genetic diversity was determined by calculating the expected heterozygosity and allelic richness. Both linkage disequilibrium and the genetic differentiation (theta) were estimated.
The proportion of polyclonal infections varied substantially by site (11% - 70%), with the expected heterozygosity ranging between 0.44 and 0.69; no haplotypes were shared between the different populations. Linkage disequilibrium was present in all populations (IAS 0.14 - 0.61) but was higher in those with fewer polyclonal infections, suggesting inbreeding and a clonal population structure. Strong population differentiation (theta = 0.45) was found and the Bayesian inference cluster analysis identified six clusters based on distinctive allele frequencies.
The P. vivax populations circulating in the Peruvian Amazon basin are genetically diverse, strongly differentiated and they have a low effective recombination rate. These results are in line with the low and clustered pattern of malaria transmission observed in the region around Iquitos city.
[Show abstract][Hide abstract] ABSTRACT: Fourteen published and three newly identified polymorphic microsatellites were used to genotype 69 Plasmodium vivax samples obtained from 39 patients detected over a period of two years who lived in a rural community of central Vietnam. All samples were polyclonal with an average expected heterozygosity of 0.86. Among the 39 patients, 16 experienced 1-5 recurrent episodes of P. vivax malaria, most of them (83%) with a different genotype profile compared with previous infections. The minimal set of microsatellites required for differentiating the genotype profiles of the recurrent infections compared with the full set of 17 microsatellites was explored. A combination of five markers was sufficient to identify all recurrent infections with an unrelated or different genotype profile compared with all previous episodes.
The American journal of tropical medicine and hygiene 02/2010; 82(2):223-7. DOI:10.4269/ajtmh.2010.09-0458 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A recently published comment on a report of Plasmodium knowlesi infections in Vietnam states that this may not accurately represent the situation in the study area because the PCR primers used may cross-hybridize with Plasmodium vivax. Nevertheless, P. knowlesi infections have been confirmed by sequencing. In addition, a neighbour-joining tree based on the 18S S-Type SSUrRNA gene shows that the Vietnamese samples clearly cluster with the P. knowlesi isolates identified in Malaysia and are distinct from the corresponding P. vivax sequences. All samples came from asymptomatic individuals who did not consult for fever during the months preceding or following the survey, indicating that asymptomatic P. knowlesi infections occur in this population, although this does not exclude the occurrence of symptomatic cases. Large-scale studies to determine the extent and the epidemiology of P. knowlesi malaria in Vietnam are further needed.
[Show abstract][Hide abstract] ABSTRACT: Considering increasing reports on human infections by Plasmodium knowlesi in Southeast Asian countries, blood samples collected during two large cross-sectional malariometric surveys carried out in a forested area of central Vietnam in 2004 and 2005 were screened for this parasite.
Blood samples collected at the 2004 survey and positive for Plasmodium malariae were randomly selected for PCR analysis detecting P. knowlesi. Blood samples collected in 2005 from the same individuals were screened again for P. knowlesi. Positive samples were confirmed by sequencing. Family members of positive cases who participated in both surveys were also screened.
Ninety-five samples with P. malariae mono- or mixed infections identified by species-specific PCR were screened for P. knowlesi. Among the five (5.2%) positive samples by PCR, three were confirmed to be P. knowlesi infections by sequencing, two young children (<5 years old) and a young man, all asymptomatic at the time of the survey and for the next six months after the survey. One of the two children was still positive one year later. No infection was found among the family members.
Plasmodium knowlesi infections in humans can be found in central Vietnam. A small child was positive for P. knowlesi in both surveys at one year interval, though it is unclear whether it was the same or a new infection.