Immunologic activation of human syncytiotrophoblast by Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Malaria during pregnancy is characterized by the sequestration of malaria-infected red blood cells (iRBC) in the intervillous spaces of the placenta, often accompanied by the infiltration of maternal mononuclear cells, causing substantial maternal and foetal/infant morbidity. The iRBC bind to receptors expressed by the syncytiotrophoblast (ST). How ST responds to this interaction remains poorly understood. Because it is known that ST is immunoactive and can respond to infectious agents, the consequences of this ST-iRBC interaction should be investigated.</p> <p>Methods</p> <p>An in vitro system was used to assess the biochemical and immunological changes induced in ST by ST-adherent iRBCs. Changes in ST mitogen-activated protein kinase (MAPK) activation were assessed by immunoblotting and mRNA expression levels of selected cytokine and chemokines in primary ST bound by iRBC were determined using real-time, reverse transcription PCR. In addition, secreted cytokine and chemokine proteins were assayed by standard ELISA, and chemotaxis of PBMC was assessed using a two-chamber assay system.</p> <p>Results</p> <p>Following iRBC/ST interaction, ST C-Jun N-terminal kinase 1 (JNK1) was activated and modest increases in the mRNA expression of TGF-β and IL-8/CXCL8 were observed. In addition, this interaction increased secretion of MIF and MIP-1α/CCL3 by ST and induced migration of PBMC towards iRBC-stimulated ST.</p> <p>Conclusion</p> <p>Results from this study provide the first evidence that ST participates in shaping the local immunological milieu and in the recruitment of maternal immune cells to the maternal blood space during placental malaria infection.</p

Field Evaluation of the Photo-induced Electron Transfer Fluorogenic Primers (PET) Real-time PCR for the Detection of Plasmodium falciparum in Tanzania.

Accurate diagnosis of malaria infections remains challenging, especially in the identification of submicroscopic infections. New molecular diagnostic tools that are inexpensive, sensitive enough to detect low-level infections and suitable in laboratory settings of resource-limited countries are required for malaria control and elimination programmes. Here the diagnostic potential of a recently developed photo-induced electron transfer fluorogenic primer (PET) real-time polymerase chain reaction (PCR) called PET-PCR was investigated. This study aimed to (i) evaluate the use of this assay as a method for the detection of both Plasmodium falciparum and other Plasmodium species infections in a developing country's diagnostic laboratory; and, (ii) determine the assay's sensitivity and specificity compared to a nested 18S rRNA PCR. Samples used in this study were obtained from a previous study conducted in the region of Iringa, Tanzania. A total of 303 samples from eight health facilities in Tanzania were utilized for this evaluation. All samples were screened using the multiplex PET-PCR assay designed to detect Plasmodium genus and P. falciparum initially in laboratory in Tanzania and then repeated at a reference laboratory at the CDC in the USA. Microscopy data was available for all the 303 samples. A subset of the samples were tested in a blinded fashion to find the sensitivity and specificity of the PET-PCR compared to the nested 18S rRNA PCR. Compared to microscopy, the PET-PCR assay was 59% more sensitive in detecting P. falciparum infections. The observed sensitivity and specificity were 100% (95% confidence interval (CI0.95) = 94-100%) and (CI0.95 = 96-100%), respectively, for the PET-PCR assay when compared to nested 18S rRNA PCR. When compared to 18S rRNA PCR, microscopy had a low sensitivity of 40% (CI0.95 = 23-61%) and specificity of 100% (CI0.95 = 96-100%). The PET-PCR results performed in the field laboratory in Tanzania were in 100% concordance with the results obtained at the reference laboratory in the USA. The PET-PCR is a new molecular diagnostic tool with similar performance characteristics as commonly used PCR methods that is less expensive, easy to use, and amiable to large scale-surveillance studies in developing country settings

Antibody responses to the merozoite surface protein-1 complex in cerebral malaria patients in India

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum </it>infection causes cerebral malaria (CM) in a subset of patients with anti-malarial treatment protecting only about 70% to 80% of patients. Why a subset of malaria patients develops CM complications, including neurological sequelae or death, is still not well understood. It is believed that host immune factors may modulate CM outcomes and there is substantial evidence that cellular immune factors, such as cytokines, play an important role in this process. In this study, the potential relationship between the antibody responses to the merozoite surface protein (MSP)-1 complex (which consists of four fragments namely: MSP-1₈₃, MSP-1₃₀, MSP-1₃₈and MSP-1₄₂), MSP-6₃₆and MSP-7₂₂and CM was investigated.</p> <p>Methods</p> <p>Peripheral blood antibody responses to recombinant antigens of the two major allelic forms of MSP-1 complex, MSP-6₃₆and MSP-7₂₂were compared between healthy subjects, mild malaria patients (MM) and CM patients residing in a malaria endemic region of central India. Total IgG and IgG subclass antibody responses were determined using ELISA method.</p> <p>Results</p> <p>The prevalence and levels of IgG and its subclasses in the plasma varied for each antigen. In general, the prevalence of total IgG, IgG1 and IgG3 was higher in the MM patients and lower in CM patients compared to healthy controls. Significantly lower levels of total IgG antibodies to the MSP-1_f38, IgG1 levels to MSP-1_d83, MSP-1₁₉and MSP-6₃₆and IgG3 levels to MSP-1_f42and MSP-7₂₂were observed in CM patients as compared to MM patients.</p> <p>Conclusion</p> <p>These results suggest that there may be some dysregulation in the generation of antibody responses to some MSP antigens in CM patients and it is worth investigating further whether perturbations of antibody responses in CM patients contribute to pathogenesis.</p

Plasma levels of angiopoietin-1 and -2 predict cerebral malaria outcome in Central India

<p>Abstract</p> <p>Background</p> <p>The mechanisms underlying the pathogenesis of cerebral malaria (CM) syndrome are not well understood. Previous studies have shown a strong association of inflammatory chemokines, apoptotic markers and angiogenic molecules with CM associated mortality. Recognizing the importance of angiopoietins (ANG) in the pathogenesis of CM, a retrospective investigation was carried out in a hospital cohort of malaria patients with <it>Plasmodium </it>infection in central India to determine if these factors could be suitable markers of CM associated severity.</p> <p>Methods</p> <p>Patients enrolled in the study were clinically characterized as healthy controls (HC), mild malaria (MM), CM survivors (CMS) and CM non-survivors (CMNS) based on their malaria status and hospital treatment outcome. Plasma ANG-1 and ANG-2 levels were assessed using sandwich ELISA. Receiver operating characteristic (ROC) curve analysis was used to calculate area under the curve (AUC) for each biomarker in order to assess predictive accuracy of individual biomarkers.</p> <p>Results</p> <p>The plasma levels of ANG-1 were lower in CMS and CMNS compared to control groups (mild malaria and healthy controls) at the time of hospital admission. On the contrary, ANG-2 levels positively correlated with malaria severity and were significantly higher in CMNS. The ratio of ANG-2/ANG-1 was highest in CMNS compared to other groups. Receiver operating characteristic curves revealed that compared to ANG-1 (AUC = 0.35), ANG-2 (AUC = 0.95) and ratio of ANG-2/ANG-1 (AUC = 0.90) were better markers to discriminate CMNS from MM cases. However, they were less specific in predicting fatal outcome amongst CM cases at the time of hospital admission.</p> <p>Conclusion</p> <p>These results suggest that at the time of admission plasma levels of ANG-2 and ratio of ANG-2/ANG-1 are clinically informative biomarkers to predict fatal CM from MM cases while they have limited usefulness in discriminating fatal CM outcomes in a pool of CM cases in endemic settings of Central India.</p

Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Diagnosis of Malaria

BACKGROUND: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. METHODOLOGY AND SIGNIFICANT FINDINGS: Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. CONCLUSION: This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs

Comparison of artemether-lumefantrine and chloroquine with and without primaquine for the treatment of Plasmodium vivax infection in Ethiopia: A randomized controlled trial

Background: Recent efforts in malaria control have resulted in great gains in reducing the burden of Plasmodium falciparum, but P. vivax has been more refractory. Its ability to form dormant liver stages confounds control and elimination efforts. To compare the efficacy and safety of primaquine regimens for radical cure, we undertook a randomized controlled trial in Ethiopia. Methods and findings: Patients with normal glucose-6-phosphate dehydrogenase status with symptomatic P. vivax mono-infection were enrolled and randomly assigned to receive either chloroquine (CQ) or artemether-lumefantrine (AL), alone or in combination with 14 d of semi-supervised primaquine (PQ) (3.5 mg/kg total). A total of 398 patients (n = 104 in the CQ arm, n = 100 in the AL arm, n = 102 in the CQ+PQ arm, and n = 92 in the AL+PQ arm) were followed for 1 y, and recurrent episodes were treated with the same treatment allocated at enrolment. The primary endpoints were the risk of P. vivax recurrence at day 28 and at day 42. The risk of recurrent P. vivax infection at day 28 was 4.0% (95% CI 1.5%–10.4%) after CQ treatment and 0% (95% CI 0%–4.0%) after CQ+PQ. The corresponding risks were 12.0% (95% CI 6.8%–20.6%) following AL alone and 2.3% (95% CI 0.6%–9.0%) following AL+PQ. On day 42, the risk was 18.7% (95% CI 12.2%–28.0%) after CQ, 1.2% (95% CI 0.2%–8.0%) after CQ+PQ, 29.9% (95% CI 21.6%–40.5%) after AL, and 5.9% (95% CI 2.4%–13.5%) after AL+PQ (overall p < 0.001). In those not prescribed PQ, the risk of recurrence by day 42 appeared greater following AL treatment than CQ treatment (HR = 1.8 [95% CI 1.0–3.2]; p = 0.059). At the end of follow-up, the incidence rate of P. vivax was 2.2 episodes/person-year for patients treated with CQ compared to 0.4 for patients treated with CQ+PQ (rate ratio: 5.1 [95% CI 2.9–9.1]; p < 0.001) and 2.3 episodes/person-year for AL compared to 0.5 for AL+PQ (rate ratio: 6.4 [95% CI 3.6–11.3]; p < 0.001). There was no difference in the occurrence of adverse events between treatment arms. The main limitations of the study were the early termination of the trial and the omission of haemoglobin measurement after day 42, resulting in an inability to estimate the cumulative risk of anaemia. Conclusions: Despite evidence of CQ-resistant P. vivax, the risk of recurrence in this study was greater following treatment with AL unless it was combined with a supervised course of PQ. PQ combined with either CQ or AL was well tolerated and reduced recurrence of vivax malaria by 5-fold at 1 y

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

A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi

Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites.The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi