Pre-elimination stage of malaria in Sri Lanka: assessing the level of hidden parasites in the population

<p>Abstract</p> <p>Background</p> <p>With the dramatic drop in the transmission of malaria in Sri Lanka in recent years, the country entered the malaria pre-elimination stage in 2008. Assessing the community prevalence of hidden malaria parasites following several years of extremely low transmission is central to the process of complete elimination. The existence of a parasite reservoir in a population free from clinical manifestations, would influence the strategy for surveillance and control towards complete elimination.</p> <p>Methods</p> <p>The prevalence of hidden parasite reservoirs in two historically malaria endemic districts, Anuradhapura and Kurunegala, previously considered as high malaria transmission areas in Sri Lanka, where peaks of transmission follow the rainy seasons was assessed. Blood samples of non-febrile individuals aged five to 55 years were collected from randomly selected areas in the two districts at community level and a questionnaire was used to collect demographic information and movement of the participants. A simple, highly sensitive nested PCR was carried out to detect both <it>Plasmodium falciparum </it>and <it>Plasmodium vivax</it>, simultaneously.</p> <p>Results</p> <p>In total, 3,023 individuals from 101 villages participated from both districts comprising mostly adults between the ages 19-55 years. Out of these, only about 1.4% of them (n = 19) could recall having had malaria during the past five years. Analysis of a subset of samples (n = 1322) from the two districts using PCR showed that none of the participants had hidden parasites.</p> <p>Discussion</p> <p>A reservoir of hidden parasites is unlikely to be a major concern or a barrier to the ongoing malaria elimination efforts in Sri Lanka. However, as very low numbers of indigenous cases are still recorded, an island-wide assessment and in particular, continued alertness and follow up action are still needed. The findings of this study indicate that any future assessments should be based on an adaptive sampling approach, involving prompt sampling of all subjects within a specified radius, whenever a malaria case is identified in a given focus.</p

Evaluation of three PCR-based diagnostic assays for detecting mixed Plasmodium infection

<p>Abstract</p> <p>Background</p> <p>One of the most commonly used molecular test for malaria diagnosis is the polymerase chain reaction (PCR)-based amplification of the 18S ribosomal DNA (rDNA) gene. Published diagnostic assays based on the 18S gene include the "gold standard" nested assay, semi-nested multiplex assay, and one tube multiplex assay. To our knowledge, no one has reported whether the two multiplex methods are better at detecting mixed <it>Plasmodium </it>infections compared to the nested assay using known quantities of DNA in experimentally mixed cocktails.</p> <p>Findings</p> <p>Here we evaluated three PCR assays (nested, semi-nested multiplex, and one-tube multiplex) for the simultaneous detection of human malaria parasites using experimentally mixed cocktails of known quantities of laboratory derived DNA. All three assays detected individual species with high sensitivity and specificity when DNA was from any one single species; however, experimentally mixed DNA cocktails with all four species present were correctly identified most consistently with the nested method. The other two methods failed to consistently identify all four species correctly, especially at lower concentrations of DNA -subclinical levels of malaria (DNA equivalent to or less than 10 parasites per microliter).</p> <p>Conclusions</p> <p>The nested PCR method remains the method of choice for the detection of mixed malaria infections and especially of sub-clinical infections. Further optimization and/or new molecular gene targets may improve the success rate of detecting multiple parasite species simultaneously using traditional PCR assays.</p

Non-variant specific antibody responses to the C-terminal region of merozoite surface protein-1 of Plasmodium falciparum (PfMSP-119) in Iranians exposed to unstable malaria transmission

<p>Abstract</p> <p>Background</p> <p>The C-terminal region of <it>Plasmodium falciparum </it>merozoite surface protein-1 (PfMSP-1₁₉) is a leading malaria vaccine candidate antigen. However, the existence of different variants of this antigen can limit efficacy of the vaccine development based on this protein. Therefore, in this study, the main objective was to define the frequency of PfMSP-1₁₉haplotypes in malaria hypoendemic region of Iran and also to analyse cross-reactive and/or variant-specific antibody responses to four PfMSP-1₁₉variant forms.</p> <p>Methods</p> <p>The PfMSP-1₁₉was genotyped in 50 infected subjects with <it>P. falciparum </it>collected during 2006-2008. Four GST-PfMSP-1₁₉variants (E/TSR/L, E/TSG/L, E/KNG/F and Q/KNG/L) were produced in <it>Escherichia coli </it>and naturally occurring IgG antibody to these proteins was evaluated in malaria patients' sera (n = 50) using ELISA. To determine the cross-reactivity of antibodies against each PfMSP-1₁₉variant in <it>P. falciparum-</it>infected human sera, an antibody depletion assay was performed in eleven corresponding patients' sera.</p> <p>Results</p> <p>Sequence data of the PfMSP-1₁₉revealed five variant forms in which the haplotypes Q/KNG/L and Q/KNG/F were predominant types and the second most frequent haplotype was E/KNG/F. In addition, the prevalence of IgG antibodies to all four PfMSP-1₁₉variant forms was equal and high (84%) among the studied patients' sera. Immunodepletion results showed that in Iranian malaria patients, Q/KNG/L variant could induce not only cross-reactive antibody responses to other PfMSP-1₁₉variants, but also could induce some specific antibodies that are not able to recognize the E/TSG/L or E/TSR/L variant forms.</p> <p>Conclusion</p> <p>The present findings demonstrated the presence of non-variant specific antibodies to PfMSP-1₁₉in Iranian falciparum malaria patients. This data suggests that polymorphism in PfMSP-1₁₉is less important and one variant of this antigen, particularly Q/KNG/L, may be sufficient to be included in PfMSP-1₁₉-based vaccine.</p

Comparative Gene Expression Profiling of P. falciparum Malaria Parasites Exposed to Three Different Histone Deacetylase Inhibitors

Histone deacetylase (HDAC) inhibitors are being intensively pursued as potential new drugs for a range of diseases, including malaria. HDAC inhibitors are also important tools for the study of epigenetic mechanisms, transcriptional control, and other important cellular processes. In this study the effects of three structurally related antimalarial HDAC inhibitors on P. falciparum malaria parasite gene expression were compared. The three hydroxamate-based compounds, trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA; Vorinostat®) and a 2-aminosuberic acid derivative (2-ASA-9), all caused profound transcriptional effects, with ∼2–21% of genes having >2-fold altered expression following 2 h exposure to the compounds. Only two genes, alpha tubulin II and a hydrolase, were up-regulated by all three compounds after 2 h exposure in all biological replicates examined. The transcriptional changes observed after 2 h exposure to HDAC inhibitors were found to be largely transitory, with only 1–5% of genes being regulated after removing the compounds and culturing for a further 2 h. Despite some structural similarity, the three inhibitors caused quite diverse transcriptional effects, possibly reflecting subtle differences in mode of action or cellular distribution. This dataset represents an important contribution to our understanding of how HDAC inhibitors act on malaria parasites and identifies alpha tubulin II as a potential transcriptional marker of HDAC inhibition in malaria parasites that may be able to be exploited for future development of HDAC inhibitors as new antimalarial agents

Effectiveness of artemether-lumefantrine provided by community health workers in under-five children with uncomplicated malaria in rural Tanzania: an open label prospective study

\ud Home-management of malaria (HMM) strategy improves early access of anti-malarial medicines to high-risk groups in remote areas of sub-Saharan Africa. However, limited data are available on the effectiveness of using artemisinin-based combination therapy (ACT) within the HMM strategy. The aim of this study was to assess the effectiveness of artemether-lumefantrine (AL), presently the most favoured ACT in Africa, in under-five children with uncomplicated Plasmodium falciparum malaria in Tanzania, when provided by community health workers (CHWs) and administered unsupervised by parents or guardians at home. An open label, single arm prospective study was conducted in two rural villages with high malaria transmission in Kibaha District, Tanzania. Children presenting to CHWs with uncomplicated fever and a positive rapid malaria diagnostic test (RDT) were provisionally enrolled and provided AL for unsupervised treatment at home. Patients with microscopy confirmed P. falciparum parasitaemia were definitely enrolled and reviewed weekly by the CHWs during 42 days. Primary outcome measure was PCR corrected parasitological cure rate by day 42, as estimated by Kaplan-Meier survival analysis. This trial is registered with ClinicalTrials.gov, number NCT00454961. A total of 244 febrile children were enrolled between March-August 2007. Two patients were lost to follow up on day 14, and one patient withdrew consent on day 21. Some 141/241 (58.5%) patients had recurrent infection during follow-up, of whom 14 had recrudescence. The PCR corrected cure rate by day 42 was 93.0% (95% CI 88.3%-95.9%). The median lumefantrine concentration was statistically significantly lower in patients with recrudescence (97 ng/mL [IQR 0-234]; n = 10) compared with reinfections (205 ng/mL [114-390]; n = 92), or no parasite reappearance (217 [121-374] ng/mL; n = 70; p ≤ 0.046). Provision of AL by CHWs for unsupervised malaria treatment at home was highly effective, which provides evidence base for scaling-up implementation of HMM with AL in Tanzania.\u

Evaluation of a novel magneto-optical method for the detection of malaria parasites

Improving the efficiency of malaria diagnosis is one of the main goals of current malaria research. We have recently developed a magneto-optical (MO) method which allows high-sensitivity detection of malaria pigment (hemozoin crystals) in blood via the magnetically induced rotational motion of the hemozoin crystals. Here, we evaluate this MO technique for the detection of Plasmodium falciparum in infected erythrocytes using in-vitro parasite cultures covering the entire intraerythrocytic life cycle. Our novel method detected parasite densities as low as approximately 40 parasites per microliter of blood (0.0008% parasitemia) at the ring stage and less than 10 parasites/microL (0.0002% parasitemia) in the case of the later stages. These limits of detection, corresponding to approximately 20 pg/microL of hemozoin produced by the parasites, exceed that of rapid diagnostic tests and compete with the threshold achievable by light microscopic observation of blood smears. The MO diagnosis requires no special training of the operator or specific reagents for parasite detection, except for an inexpensive lysis solution to release intracellular hemozoin. The devices can be designed to a portable format for clinical and in-field tests. Besides testing its diagnostic performance, we also applied the MO technique to investigate the change in hemozoin concentration during parasite maturation. Our preliminary data indicate that this method may offer an efficient tool to determine the amount of hemozoin produced by the different parasite stages in synchronized cultures. Hence, it could eventually be used for testing the susceptibility of parasites to antimalarial drugs