Katherine Torres

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.

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.

Anti-Plasmodium falciparum invasion ligand antibodies in a low malaria transmission region, Loreto, Peru.

BackgroundErythrocyte invasion by Plasmodium falciparum is a complex process that involves two families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins. Antibodies that inhibit merozoite attachment and invasion are believed to be important in mediating naturally acquired immunity and immunity generated by parasite blood stage vaccine candidates. The hypotheses tested in this study were 1) that antibody responses against specific P. falciparum invasion ligands (EBL and PfRh) differ between symptomatic and asymptomatic individuals living in the low-transmission region of the Peruvian Amazon and 2), such antibody responses might have an association, either direct or indirect, with clinical immunity observed in asymptomatically parasitaemic individuals.MethodsELISA was used to assess antibody responses (IgG, IgG1 and IgG3) against recombinant P. falciparum invasion ligands of the EBL (EBA-175, EBA-181, EBA-140) and PfRh families (PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5) in 45 individuals infected with P. falciparum from Peruvian Amazon. Individuals were classified as having symptomatic malaria (N=37) or asymptomatic infection (N=8).ResultsAntibody responses against both EBL and PfRh family proteins were significantly higher in asymptomatic compared to symptomatic individuals, demonstrating an association with clinical immunity. Significant differences in the total IgG responses were observed with EBA-175, EBA-181, PfRh2b, and MSP119 (as a control). IgG1 responses against EBA-181, PfRh2a and PfRh2b were significantly higher in the asymptomatic individuals. Total IgG antibody responses against PfRh1, PfRh2a, PfRh2b, PfRh5, EBA-175, EBA-181 and MSP119 proteins were negatively correlated with level of parasitaemia. IgG1 responses against EBA-181, PfRh2a and PfRh2b and IgG3 response for PfRh2a were also negatively correlated with parasitaemia.ConclusionsThese data suggest that falciparum malaria patients who develop clinical immunity (asymptomatic parasitaemia) in a low transmission setting such as the Peruvian Amazon have antibody responses to defined P. falciparum invasion ligand proteins higher than those found in symptomatic (non-immune) patients. While these findings will have to be confirmed by larger studies, these results are consistent with a potential role for one or more of these invasion ligands as a component of an anti-P. falciparum vaccine in low-transmission malaria-endemic regions.

Antibody response dynamics to the Plasmodium falciparum conserved vaccine candidate antigen, merozoite surface protein-1 C-terminal 19kD (MSP1-19kD), in Peruvians exposed to hypoendemic malaria transmission.

BackgroundIn high-transmission areas, developing immunity to symptomatic Plasmodium falciparum infections requires 2–10 years of uninterrupted exposure. Delayed malaria-immunity has been attributed to difficult-to-develop and then short-lived antibody responses.MethodsIn a study area with <0.5 P. falciparum infections/person/year, antibody responses to the MSP1-19kD antigen were evaluated and associations with P. falciparum infections in children and adults. In months surrounding and during the malaria seasons of 2003–2004, 1,772 participants received ≥6 active visits in one study-year. Community-wide surveys were conducted at the beginning and end of each malaria season, and weekly active visits were completed for randomly-selected individuals each month. There were 79 P. falciparum infections with serum samples collected during and approximately one month before and after infection. Anti-MSP1-19kD IgG levels were measured by ELISA.ResultsThe infection prevalence during February-July was similar in children (0.02–0.12 infections/person/month) and adults (0.03–0.14 infections/person/month) and was negligible in the four-month dry season. In children and adults, the seroprevalence was maintained in the beginning (children = 28.9%, adults = 61.8%) versus ending malaria-season community survey (children = 26.7%, adults = 64.6%). Despite the four-month non-transmission season, the IgG levels in Plasmodium-negative adults were similar to P. falciparum-positive adults. Although children frequently responded upon infection, the transition from a negative/low level before infection to a high level during/after infection was slower in children. Adults and children IgG-positive before infection had reduced symptoms and parasite density.ConclusionIndividuals in low transmission areas can rapidly develop and maintain αMSP1-19kD IgG responses for >4 months, unlike responses reported in high transmission study areas. A greater immune capacity might contribute to the frequent asymptomatic P. falciparum infections in this Peruvian population.

Relationship of regulatory T cells to Plasmodium falciparum malaria symptomatology in a hypoendemic region

BackgroundPrevious data have suggested that regulatory T cells (Tregs) balance protective immune responses with immune mediated pathology in malaria. This study aimed to determine to test the hypothesis that Treg proportions or absolute levels are associated with parasitaemia and malaria symptoms.MethodsTreg cells were quantified by flow cytometry as CD4+ CD25+, Foxp3+, CD127low T cells. Three patient groups were assessed: patients with symptomatic Plasmodium falciparum malaria (S), subjects with asymptomatic P. falciparum parasitaemia (AS) and uninfected control individuals (C).ResultsS, AS and C groups had similar absolute numbers and percentage of Tregs (3.9%, 3.5% and 3.5% respectively). Levels of parasitaemia were not associated with Treg percentage (p = 0.47).ConclusionNeither relative nor absolute regulatory T cell numbers were found to be associated with malaria-related symptomatology in this study. Immune mechanisms other than Tregs are likely to be responsible for the state of asymptomatic P. falciparum parasitaemia in the Peruvian Amazon; but further study to explore these mechanisms is needed.

Genome-Level Determination of Plasmodium falciparum Blood-Stage Targets of Malarial Clinical Immunity in the Peruvian Amazon

BACKGROUND Persons with blood-stage Plasmodium falciparum parasitemia in the absence of symptoms are considered to be clinically immune. We hypothesized that asymptomatic subjects with P. falciparum parasitemia would differentially recognize a subset of P. falciparum proteins on a genomic scale. METHODS AND FINDINGS Compared with symptomatic subjects, sera from clinically immune, asymptomatically infected individuals differentially recognized 51 P. falciparum proteins, including the established vaccine candidate PfMSP1. Novel, hitherto unstudied hypothetical proteins and other proteins not previously recognized as potential vaccine candidates were also differentially recognized. Genes encoding the proteins differentially recognized by the Peruvian clinically immune individuals exhibited a significant enrichment of nonsynonymous nucleotide variation, an observation consistent with these genes undergoing immune selection. CONCLUSIONS A limited set of P. falciparum protein antigens was associated with the development of naturally acquired clinical immunity in the low-transmission setting of the Peruvian Amazon. These results imply that, even in a low-transmission setting, an asexual blood-stage vaccine designed to reduce clinical malaria symptoms will likely need to contain large numbers of often-polymorphic proteins, a finding at odds with many current efforts in the design of vaccines against asexual blood-stage P. falciparum.

Brucella melitensis T Cell Epitope Recognition in Humans with Brucellosis in Peru

ABSTRACT Brucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic, and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes that might provide new approaches to classifying and prognosticating B. melitensis infection have ever been delineated. Twenty-seven pools of 500 major histocompatibility complex class II (MHC-II) restricted peptides were created by computational prediction of promiscuous MHC-II CD4+ T cell derived from the top 50 proteins recognized by IgG in human sera on a genome level B. melitensis protein microarray. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) enzyme-linked immunospot (ELISPOT) analyses were used to quantify and compare Th1 and Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (n = 9) and from patients who relapsed (n = 5). Four peptide epitopes derived from 3 B. melitensis proteins (BMEI 1330, a DegP/HtrA protease; BMEII 0029, type IV secretion system component VirB5; and BMEII 0691, a predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-γ ELISPOT responses in both acute-infection and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant. These experiments are the first to systematically identify B. melitensis MHC-II-restricted CD4+ T cell epitopes recognized by the human immune response, with the potential for new approaches to brucellosis diagnostics and understanding the immunopathogenesis related to this intracellular pathogen.