Dionicia Gamboa

A Large Proportion of P. falciparum Isolates in the Amazon Region of Peru Lack pfhrp2 and pfhrp3: Implications for Malaria Rapid Diagnostic Tests

Background Malaria rapid diagnostic tests (RDTs) offer significant potential to improve the diagnosis of malaria, and are playing an increasing role in malaria case management, control and elimination. Peru, along with other South American countries, is moving to introduce malaria RDTs as components of malaria control programmes supported by the Global Fund for AIDS, TB and malaria. The selection of the most suitable malaria RDTs is critical to the success of the programmes. Methods Eight of nine microscopy positive P. falciparum samples collected in Iquitos, Peru tested negative or weak positive using HRP2-detecting RDTs. These samples were tested for the presence of pfhrp2 and pfhrp3 and their flanking genes by PCR, as well as the presence of HRP proteins by ELISA. To investigate for geographic extent of HRP-deleted parasites and their temporal occurrence a retrospective study was undertaken on 148 microscopy positive P. falciparum samples collected in different areas of the Amazon region of Peru. Findings Eight of the nine isolates lacked the pfhrp2 and/or pfhrp3 genes and one or both flanking genes, and the absence of HRP was confirmed by ELISA. The retrospective study showed that 61 (41%) and 103 (70%) of the 148 samples lacked the pfhrp2 or pfhrp3 genes respectively, with 32 (21.6%) samples lacking both hrp genes. Conclusions This is the first documentation of P. falciparum field isolates lacking pfhrp2 and/or pfhrp3. The high frequency and wide distribution of different parasites lacking pfhrp2 and/or pfhrp3 in widely dispersed areas in the Peruvian Amazon implies that malaria RDTs targeting HRP2 will fail to detect a high proportion of P. falciparum in malaria-endemic areas of Peru and should not be used. RDTs detecting parasite LDH or aldolase and quality microscopy should be use for malaria diagnosis in this region. There is an urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighbouring countries.

Amazonian malaria: asymptomatic human reservoirs, diagnostic challenges, environmentally driven changes in mosquito vector populations, and the mandate for sustainable control strategies.

Across the Americas and the Caribbean, nearly 561,000 slide-confirmed malaria infections were reported officially in 2008. The nine Amazonian countries accounted for 89% of these infections; Brazil and Peru alone contributed 56% and 7% of them, respectively. Local populations of the relatively neglected parasite Plasmodium vivax, which currently accounts for 77% of the regional malaria burden, are extremely diverse genetically and geographically structured. At a time when malaria elimination is placed on the public health agenda of several endemic countries, it remains unclear why malaria proved so difficult to control in areas of relatively low levels of transmission such as the Amazon Basin. We hypothesize that asymptomatic parasite carriage and massive environmental changes that affect vector abundance and behavior are major contributors to malaria transmission in epidemiologically diverse areas across the Amazon Basin. Here we review available data supporting this hypothesis and discuss their implications for current and future malaria intervention policies in the region. Given that locally generated scientific evidence is urgently required to support malaria control interventions in Amazonia, we briefly describe the aims of our current field-oriented malaria research in rural villages and gold-mining enclaves in Peru and a recently opened agricultural settlement in Brazil.

Rapid Diagnostic Tests for Malaria Diagnosis in the Peruvian Amazon: Impact of pfhrp2 Gene Deletions and Cross-Reactions

Background In the Peruvian Amazon, Plasmodium falciparum and Plasmodium vivax malaria are endemic in rural areas, where microscopy is not available. Malaria rapid diagnostic tests (RDTs) provide quick and accurate diagnosis. However, pfhrp2 gene deletions may limit the use of histidine-rich protein-2 (PfHRP2) detecting RDTs. Further, cross-reactions of P. falciparum with P. vivax-specific test lines and vice versa may impair diagnostic specificity. Methods Thirteen RDT products were evaluated on 179 prospectively collected malaria positive samples. Species diagnosis was performed by microscopy and confirmed by PCR. Pfhrp2 gene deletions were assessed by PCR. Results Sensitivity for P. falciparum diagnosis was lower for PfHRP2 compared to P. falciparum-specific Plasmodium lactate dehydrogenase (Pf-pLDH)- detecting RDTs (71.6% vs. 98.7%, p<0.001). Most (19/21) false negative PfHRP2 results were associated with pfhrp2 gene deletions (25.7% of 74 P. falciparum samples). Diagnostic sensitivity for P. vivax (101 samples) was excellent, except for two products. In 10/12 P. vivax-detecting RDT products, cross-reactions with the PfHRP2 or Pf-pLDH line occurred at a median frequency of 2.5% (range 0%–10.9%) of P. vivax samples assessed. In two RDT products, two and one P. falciparum samples respectively cross-reacted with the Pv-pLDH line. Two Pf-pLDH/pan-pLDH-detecting RDTs showed excellent sensitivity with few (1.0%) cross-reactions but showed faint Pf-pLDH lines in 24.7% and 38.9% of P. falciparum samples. Conclusion PfHRP2-detecting RDTs are not suitable in the Peruvian Amazon due to pfhrp2 gene deletions. Two Pf-pLDH-detecting RDTs performed excellently and are promising RDTs for this region although faint test lines are of concern.

Multiple independent introductions of Plasmodium falciparum in South America

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence—archeological and genetic—suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Multilocus genotyping reveals high heterogeneity and strong local population structure of the Plasmodium vivax population in the Peruvian Amazon

BackgroundPeru 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.MethodsPlasmodium 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 (θ) were estimated.ResultsThe 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 (θ = 0.45) was found and the Bayesian inference cluster analysis identified six clusters based on distinctive allele frequencies.ConclusionThe 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.

Adherence to 7-Day Primaquine Treatment for the Radical Cure of P. vivax in the Peruvian Amazon

Despite being free of charge, treatment adherence to 7-day primaquine for the radical cure of Plasmodium vivax was estimated at 62.2% among patients along the Iquitos-Nauta road in the Peruvian Amazon. The principal reason for non-adherence was the perceived adverse effects related to local humoral illness conceptions that hold that malaria produces a hot state of body, which is further aggravated by the characteristically hot medical treatment. Notably, patients were willing to adhere to the first 3 days of treatment during which symptoms are most apparent and include the characteristic chills. Nevertheless, as symptoms abate, the perceived aggravating characteristics of the medication outweigh the perceived advantages of treatment adherence. Improving community awareness about the role of primaquine to prevent further malaria transmission and fostering a realistic system of direct observed treatment intake, organized at community level, can be expected to improve adherence to the radical cure of P. vivax in this area.

Plasmodium vivax Sub-Patent Infections after Radical Treatment Are Common in Peruvian Patients: Results of a 1-Year Prospective Cohort Study

Background 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. Methodology 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. Results 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 (IAs = 0.29, p<1.10−4) that remained also when analyzing only the unique haplotypes, suggesting common inbreeding. Conclusion 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.

Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.

Isolation and molecular identification of Leishmania ( Viannia ) peruviana from naturally infected Lutzomyia peruensis (Diptera: Psychodidae) in the Peruvian Andes

Leishmania (Viannia) peruviana was isolated from 1/75 Lutzomyia peruensis captured during May 2006 in an endemic cutaneous leishmaniasis region of the Peruvian Andes (Chaute, Huarochiri, Lima, Peru). Sand fly gut with promastigotes was inoculated into a hamster and the remaining body was fixed in ethanol. L. (Viannia) sp. was determined by polymerase chain reaction (PCR), and Leishmania species through molecular genotyping by PCR-restriction fragment length polymorphism analyses targeting the genes cpb and hsp70, resulting L. (V.) peruviana. The infected sand fly appeared 15 days after the rains finished, time expected and useful real time data for interventions when transmission is occurring.

Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from 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.