We evaluated the efficacy of three primaquine (PQ) regimes to prevent relapses with Plasmodium vivax through an open-label randomized trial in Loreto, Peru. Vivax monoinfections were treated with chloroquine for 3 days and PQ in three different regimes: 0.5 mg/kg per day for 5 days (150 mg total), 0.5 mg/kg per day for 7 days (210 mg total), or 0.25 mg/kg per day for 14 days (210 mg total). Biweekly fever assessments and bimonthly thick smears were taken for 210 days. Recurrences after 35 days were considered relapses. One hundred eighty cases were enrolled in each group; 90% of cases completed follow-up. There were no group-related differences in age, sex, or parasitemia. Relapse rates were similar in the 7- and 14-day regimes (16/156 = 10.3% and 22/162 = 13.6%, P = 0.361) and higher in the 5-day group (48/169 = 28.4%, P < 0.001 and P = 0.001, respectively). The 7-day PQ regimen used in Peru is as efficacious as the recommended 14-day regimen and superior to 5 treatment days.
Plasmodium vivax is a predominant species of malaria in parts of South America and there is increasing resistance to drugs to treat infections by P. vivax. The existence of latent hypnozoites further complicates the ability to classify recurrent infections as treatment failures due to relapse, recrudescence of hyponozoites or re-infections. Antigen loci are putatively under natural selection and may not be an optimal molecular marker to define parasite haplotypes in paired samples. Putatively neutral microsatellite loci, however, offer an assessment of neutral haplotypes. The objective here was to assess the utility of neutral microsatellite loci to reconcile cases of recurrent parasitaemia in Amazonian P. vivax populations in Peru.
The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times.
Plasmodium vivax is the most prevalent human malaria parasite in the Americas. Previous studies have contrasted the genetic diversity of parasite populations in the Americas with those in Asia and Oceania, concluding that New World populations exhibit low genetic diversity consistent with a recent introduction. Here we used an expanded sample of complete mitochondrial genome sequences to investigate the diversity of P. vivax in the Americas as well as in other continental populations. We show that the diversity of P. vivax in the Americas is comparable to that in Asia and Oceania, and we identify several divergent clades circulating in South America that may have resulted from independent introductions. In particular, we show that several haplotypes sampled in Venezuela and northeastern Brazil belong to a clade that diverged from the other P. vivax lineages at least 30,000 years ago, albeit not necessarily in the Americas. We propose that, unlike in Asia where human migration increases local genetic diversity, the combined effects of the geographical structure and the low incidence of vivax malaria in the Americas has resulted in patterns of low local but high regional genetic diversity. This could explain previous views that P. vivax in the Americas has low genetic diversity because these were based on studies carried out in limited areas. Further elucidation of the complex geographical pattern of P. vivax variation will be important both for diversity assessments of genes encoding candidate vaccine antigens and in the formulation of control and surveillance measures aimed at malaria elimination.
The merozoite surface protein-9 (MSP-9) has been considered a target for an anti-malarial vaccine since it is one of many proteins involved in the erythrocyte invasion, a critical step in the parasite life cycle. Orthologs encoding this antigen have been found in all known species of Plasmodium parasitic to primates. In order to characterize and investigate the extent and maintenance of MSP-9 genetic diversity, we analyzed DNA sequences of the following malaria parasite species: Plasmodium falciparum, Plasmodium reichenowi, Plasmodium chabaudi, Plasmodium yoelii, Plasmodium berghei, Plasmodium coatneyi, Plasmodium gonderi, Plasmodium knowlesi, Plasmodium inui, Plasmodium simiovale, Plasmodium fieldi, Plasmodium cynomolgi and Plasmodium vivax and evaluated the signature of natural selection in all MSP-9 orthologs. Our findings suggest that the gene encoding MSP-9 is under purifying selection in P. vivax and closely related species. We further explored how selection affected different regions of MSP-9 by comparing the polymorphisms in P. vivax and P. falciparum, and found contrasting patterns between these two species that suggest differences in functional constraints. This observation implies that the MSP-9 orthologs in human parasites may interact differently with the host immune response. Thus, studies carried out in one species cannot be directly translated into the other.
Rapidly characterizing the amplitude and variability in transmissibility of novel human influenza strains as they emerge is a key public health priority. However, comparison of early estimates of the basic reproduction number during the 2009 pandemic were challenging because of inconsistent data sources and methods. Here, we define and analyze influenza-like-illness (ILI) case data from 2009-2010 for the 50 largest spatially distinct US military installations (military population defined by zip code, MPZ). We used publicly available data from non-military sources to show that patterns of ILI incidence in many of these MPZs closely followed the pattern of their enclosing civilian population. After characterizing the broad patterns of incidence (e.g. single-peak, double-peak), we defined a parsimonious SIR-like model with two possible values for intrinsic transmissibility across three epochs. We fitted the parameters of this model to data from all 50 MPZs, finding them to be reasonably well clustered with a median (mean) value of 1.39 (1.57) and standard deviation of 0.41. An increasing temporal trend in transmissibility ([Formula: see text], p-value: 0.013) during the period of our study was robust to the removal of high transmissibility outliers and to the removal of the smaller 20 MPZs. Our results demonstrate the utility of rapidly available - and consistent - data from multiple populations.
More than half of the volume of our Universe is occupied by cosmic voids. The lensing magnification effect from those underdense regions is generally thought to give a small dimming contribution: objects on the far side of a void are supposed to be observed as slightly smaller than if the void were not there, which together with conservation of surface brightness implies net reduction in photons received. This is predicted by the usual weak lensing integral of the density contrast along the line of sight. We show that this standard effect is swamped at low redshifts by a relativistic Doppler term that is typically neglected. Contrary to the usual expectation, objects on the far side of a void are brighter than they would be otherwise. Thus the local dynamics of matter in and near the void is crucial and is only captured by the full relativistic lensing convergence. There are also significant nonlinear corrections to the relativistic linear theory, which we show actually underpredicts the effect. We use exact solutions to estimate that these can be more than 20% for deep voids. This remains an important source of systematic errors for weak lensing density reconstruction in galaxy surveys and for supernovae observations, and may be the cause of the reported extra scatter of field supernovae located on the edge of voids compared to those in clusters.
In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.
Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradications successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999-2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006-2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase.
Capacity-building initiatives related to public health are defined as developing laboratory infrastructure, strengthening host-country disease surveillance initiatives, transferring technical expertise and training personnel. These initiatives represented a major piece of the Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) contributions to worldwide emerging infectious disease (EID) surveillance and response. Capacity-building initiatives were undertaken with over 80 local and regional Ministries of Health, Agriculture and Defense, as well as other government entities and institutions worldwide. The efforts supported at least 52 national influenza centers and other country-specific influenza, regional and U.S.-based EID reference laboratories (44 civilian, eight military) in 46 countries worldwide. Equally important, reference testing, laboratory infrastructure and equipment support was provided to over 500 field sites in 74 countries worldwide from October 2008 to September 2009. These activities allowed countries to better meet the milestones of implementation of the 2005 International Health Regulations and complemented many initiatives undertaken by other U.S. government agencies, such as the U.S. Department of Health and Human Services, the U.S. Agency for International Development and the U.S. Department of State.
Here, we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos region using genome scanning, a microarray-based technique that delineates the majority of single-base changes, indels, and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon bears a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes, indicating mutations arising during self-mating or mitotic replication. The data also reveal that one or two meioses separate different isolates, showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pairwise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species, but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase of clindamycin EC(50) in strains harboring one of these mutations. This evidence of clindamycin-resistant parasites in the Amazon suggests that a shift should be made in health policy away from quinine + clindamycin therapy for malaria in pregnant women and infants, and that the development of new lincosamide antibiotics for malaria should be reconsidered.
Several single nucleotide polymorphisms (SNPs) have been linked to antimalarial drug resistance in Plasmodium falciparum. However, standard polymerase chain reaction (PCR) methods to detect these polymorphisms are unable to detect SNPs in variants representing < 20% of the parasites in a mixed infection, nor can they detect polymorphisms at nearby loci. Here we use heteroduplex tracking assays (HTAs) to analyze dhps540 mutations in 96 samples from Peru and pfcrt76 mutations in 70 samples from China. All samples had been previously analyzed by standard PCR. We detected drug-resistant minority variants and two novel non-synonymous pfcrt mutations in China. In Peru, we found no drug-resistant minority variants and a synonymous mutation in dhps. Thus, even in regions of low malaria transmission, HTA assays are more informative than PCR with agarose gel electrophoresis.
Artemisinin-based combination therapy (ACT) is recommended as a means of prolonging the effectiveness of first-line malaria treatment regimens. Different brands of mefloquine (MQ) have been reported to be non-bioequivalent; this could result in sub-therapeutic levels of mefloquine with decreased efficacy. In 2002, mefloquine-artesunate (MQ-AS) combination therapy was adopted as the first-line treatment for uncomplicated Plasmodium falciparum malaria in the Amazon region of Peru. Although MQ resistance has yet to be reported from the Peruvian Amazon, it has been reported from other countries in the Amazon Region. Therefore, continuous monitoring is warranted to ensure that the first-line therapy remains efficacious. This study examines the in vivo efficacy and pharmacokinetic parameters through Day 56 of three commercial formulations of MQ (Lariam, Mephaquin, and Mefloquina-AC Farma) given in combination with artesunate.
All New World Leishmania species can cause cutaneous lesions, while only Leishmania (Viannia) braziliensis has been associated with mucosal metastases. Multilocus enzyme electrophoresis (MLEE) is the optimal standard for species identification but is slow and costly. New methods for species identification are needed to ensure proper identification and therapy. The coding regions of four metabolic enzyme markers in the MLEE typing method: mannose phosphate isomerase (MPI), malate dehydrogenase (MDH), glucose-6-phosphate isomerase (GPI), and 6-phosphogluconate dehydrogenase (6PGD), were analysed from seven species of New World Leishmania isolated from patients with either cutaneous or mucosal lesions to identify specific genetic polymorphisms responsible for the phenotypic variations observed in the MLEE typing scheme. We identified species-specific polymorphisms and determined that a combination of sequencing of the mpi and 6pgd genes was sufficient to differentiate among seven closely related species of New World Leishmania and among isolates of L. braziliensis shown previously to have atypical MLEE patterns. When DNA isolated from 10 cutaneous lesion biopsies were evaluated, the sequence typing method was 100% concordant with the published MLEE/monoclonal antibody identification methods. The identification of species-specific polymorphisms can be used to design a DNA-based test with greater discriminatory power that requires shorter identification times. When the causative agent of the disease is L. braziliensis, this method ensures correct species identification, even when the agent is a genetic variant. Proper identification could facilitate adequate treatment, preventing the onset of the disfiguring mucosal form of the disease.
Sulfadoxine-pyrimethamine was a common first line drug therapy to treat uncomplicated falciparum malaria, but increasing therapeutic failures associated with the development of significant levels of resistance worldwide has prompted change to alternative treatment regimes in many national malaria control programs. METHODOLOGY AND FINDING: We conducted an in vivo therapeutic efficacy trial of sulfadoxine-pyrimethamine at two locations in the Peruvian Amazon enrolling 99 patients of which, 86 patients completed the protocol specified 28 day follow up. Our objective was to correlate the presence of polymorphisms in P. falciparum dihydrofolate reductase and dihydropteroate synthase to in vitro parasite susceptibility to sulfadoxine and pyrimethamine and to in vivo treatment outcomes. Inhibitory concentration 50 values of isolates increased with numbers of mutations (single [108N], sextuplet [BR/51I/108N/164L and 437G/581G]) and septuplet (BR/51I/108N/164L and 437G/540E/581G) with geometric means of 76 nM (35-166 nM), 582 nM (49-6890- nM) and 4909 (3575-6741 nM) nM for sulfadoxine and 33 nM (22-51 nM), 81 nM (19-345 nM), and 215 nM (176-262 nM) for pyrimethamine. A single mutation present in the isolate obtained at the time of enrollment from either dihydrofolate reductase (164L) or dihydropteroate synthase (540E) predicted treatment failure as well as any other single gene alone or in combination. Patients with the dihydrofolate reductase 164L mutation were 3.6 times as likely to be treatment failures [failures 85.4% (164L) vs 23.7% (I164); relative risk = 3.61; 95% CI: 2.14 - 6.64] while patients with the dihydropteroate synthase 540E were 2.6 times as likely to fail treatment (96.7% (540E) vs 37.5% (K540); relative risk = 2.58; 95% CI: 1.88 - 3.73). Patients with both dihydrofolate reductase 164L and dihydropteroate synthase 540E mutations were 4.1 times as likely to be treatment failures [96.7% vs 23.7%; RR = 4.08; 95% CI: 2.45 - 7.46] compared to patients having both wild forms (I164 and K540).
Monitoring changes in the frequencies of drug-resistant and -sensitive genotypes can facilitate in vivo clinical trials to assess the efficacy of drugs before complete failure occurs. Peru changed its national treatment policy for uncomplicated malaria to artesunate (ART)-plus-mefloquine (MQ) combination therapy in the Amazon basin in 2001. We genotyped isolates collected in 1999 and isolates collected in 2006 to 2007 for mutations in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes, multidrug resistance gene 1 (Pfmdr-1), the chloroquine (CQ) resistance transporter gene (Pfcrt), and the Ca(2+) ATPase gene (PfATP6); these have been shown to be involved in resistance to sulfadoxine-pyrimethamine (SP), MQ, CQ, and possibly ART, respectively. Microsatellite haplotypes around the Pfdhfr, Pfdhps, Pfcrt, and Pfmdr-1 loci were also determined. There was a significant decline in the highly SP resistant Pfdhfr and Pfdhps genotypes from 1999 to 2006. In contrast, a CQ-resistant Pfcrt genotype increased in frequency during the same period. Among five different Pfmdr-1 allelic forms noted in 1999, two genotypes increased in frequency while one genotype decreased by 2006. We also noted previously undescribed polymorphisms in the PfATP6 gene as well as an increase in the frequency of a deletion mutant during this period. In addition, microsatellite analysis revealed that the resistant Pfdhfr, Pfdhps, and Pfcrt genotypes have each evolved from a single founder haplotype, while Pfmdr-1 genotypes have evolved from at least two independent haplotypes. Importantly, this study demonstrates that the Peruvian triple mutant Pfdhps genotypes are very similar to those found in other parts of South America.
The widespread use of primaquine (PQ) and chloroquine (CQ), together, may be responsible for the relatively few, isolated cases of chloroquine-resistant P. vivax (CQRPV) that have been reported from South America. We report here a case of P. vivax from the Amazon Basin of Peru that recurred against normally therapeutic blood levels of CQ. Four out of 540 patients treated with combination CQ and PQ had a symptomatic recurrence of P. vivax parasitemia within 35 days of treatment initiation, possibly indicating CQ failure. Whole blood total CQ level for one of these four subjects was 95?ng/ml on the day of recurrence. Based on published criteria that delineate CQRPV as a P. vivax parasitemia, either recrudescence or relapse, that appears against CQ blood levels >100?ng/mL, we document the occurrence of a P. vivax strain in Peru that had unusually high tolerance to the synergistic combination therapy of CQ + PQ that normally works quite well.
A major concern in malaria vaccine development is genetic polymorphisms typically observed among Plasmodium isolates in different geographical areas across the world. Highly polymorphic regions have been observed in Plasmodium falciparum and Plasmodium vivax antigenic surface proteins such as Circumsporozoite protein (CSP), Duffy-binding protein (DBP), Merozoite surface protein-1 (MSP-1), Apical membrane antigen-1 (AMA-1) and Thrombospondin related anonymous protein (TRAP).
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