C5a Enhances Dysregulated Inflammatory and Angiogenic Responses to Malaria In Vitro: Potential Implications for Placental Malaria

Placental malaria (PM) is a leading cause of maternal and infant mortality. Although the accumulation of parasitized erythrocytes (PEs) and monocytes within the placenta is thought to contribute to the pathophysiology of PM, the molecular mechanisms underlying PM remain unclear. Based on the hypothesis that excessive complement activation may contribute to PM, in particular generation of the potent inflammatory peptide C5a, we investigated the role of C5a in the pathogenesis of PM in vitro and in vivo.Using primary human monocytes, the interaction between C5a and malaria in vitro was assessed. CSA- and CD36-binding PEs induced activation of C5 in the presence of human serum. Plasmodium falciparum GPI (pfGPI) enhanced C5a receptor expression (CD88) on monocytes, and the co-incubation of monocytes with C5a and pfGPI resulted in the synergistic induction of cytokines (IL-6, TNF, IL-1beta, and IL-10), chemokines (IL-8, MCP-1, MIP1alpha, MIP1beta) and the anti-angiogenic factor sFlt-1 in a time and dose-dependent manner. This dysregulated response was abrogated by C5a receptor blockade. To assess the potential role of C5a in PM, C5a plasma levels were measured in malaria-exposed primigravid women in western Kenya. Compared to pregnant women without malaria, C5a levels were significantly elevated in women with PM.These results suggest that C5a may contribute to the pathogenesis of PM by inducing dysregulated inflammatory and angiogenic responses that impair placental function

A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination.

BACKGROUND: To detect all malaria infections in elimination settings sensitive, high throughput and field deployable diagnostic tools are required. Loop-mediated isothermal amplification (LAMP) represents a possible field-applicable molecular diagnostic tool. However, current LAMP platforms are limited by their capacity for high throughput. METHODS: A high-throughput LAMP (HtLAMP) platform amplifying mitochondrial targets using a 96-well microtitre plate platform, processing 85 samples and 11 controls, using hydroxynaphtholblue as a colourimetric indicator was optimized for the detection of malaria parasites. Objective confirmation of visually detectable colour change results was made using a spectrophotometer. A dilution series of laboratory-cultured 3D7 Plasmodium falciparum parasites was used to determine the limit of detection of the HtLAMP assay, using P. falciparum (HtLAMP-Pf) and Plasmodium genus (HtLAMP-Pg) primers, on whole blood and filter paper, and using different DNA extraction protocols. The diagnostic accuracy of HtLAMP was validated using clinical samples from Papua New Guinea, Malaysia, Ghana and The Gambia and its field applicability was evaluated in Kota Marudu district hospital, Sabah, Malaysia. RESULTS: The HtLAMP assay proved to be a simple method generating a visually-detectable blue and purple colour change that could be objectively confirmed in a spectrophotometer at a wavelength of 600 nm. When compared with PCR, overall HtLAMP-Pg had a sensitivity of 98 % (n = 260/266, 95 % CI 95-99) and specificity 83 % (n = 15/18, 95 % CI 59-96). HtLAMP-Pf had a sensitivity of 97 % (n = 124/128, 95 % CI 92-99) and specificity of 96 % (n = 151/157, 95 % CI 92-99). A validation study in a regional hospital laboratory demonstrated ease of performance and interpretation of the HtLAMP assay. HtLAMP-Pf performed in this field setting had a sensitivity of 100 % (n = 17/17, 95 % CI 80-100) and specificity of 95 % (n = 123/128, 95 % CI 90-98) compared with multiplex PCR. HtLAMP-Pf also performed well on filter paper samples from asymptomatic Ghanaian children with a sensitivity of 88 % (n = 23/25, 95 % CI 69-97). CONCLUSION: This colourimetric HtLAMP assay holds much promise as a field applicable molecular diagnostic tool for the purpose of malaria elimination

Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1

Background: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reversetranscription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. Methodology/Significant Findings: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. Conclusion: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides

Maternal Malaria Induces a Procoagulant and Antifibrinolytic State That Is Embryotoxic but Responsive to Anticoagulant Therapy

Low birth weight and fetal loss are commonly attributed to malaria in endemic areas, but the cellular and molecular mechanisms that underlie these poor birth outcomes are incompletely understood. Increasing evidence suggests that dysregulated hemostasis is important in malaria pathogenesis, but its role in placental malaria (PM), characterized by intervillous sequestration of Plasmodium falciparum, proinflammatory responses, and excessive fibrin deposition is not known. To address this question, markers of coagulation and fibrinolysis were assessed in placentae from malaria-exposed primigravid women. PM was associated with significantly elevated placental monocyte and proinflammatory marker levels, enhanced perivillous fibrin deposition, and increased markers of activated coagulation and suppressed fibrinolysis in placental plasma. Submicroscopic PM was not proinflammatory but tended to be procoagulant and antifibrinolytic. Birth weight trended downward in association with placental parasitemia and high fibrin score. To directly assess the importance of coagulation in malaria-induced compromise of pregnancy, Plasmodium chabaudi AS-infected pregnant C57BL/6 mice were treated with the anticoagulant, low molecular weight heparin. Treatment rescued pregnancy at midgestation, with substantially decreased rates of active abortion and reduced placental and embryonic hemorrhage and necrosis relative to untreated animals. Together, the results suggest that dysregulated hemostasis may represent a novel therapeutic target in malaria-compromised pregnancies

Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform: A Potential Novel Tool for Malaria Elimination.

INTRODUCTION: Plasmodium vivax malaria has a wide geographic distribution and poses challenges to malaria elimination that are likely to be greater than those of P. falciparum. Diagnostic tools for P. vivax infection in non-reference laboratory settings are limited to microscopy and rapid diagnostic tests but these are unreliable at low parasitemia. The development and validation of a high-throughput and sensitive assay for P. vivax is a priority. METHODS: A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia. RESULTS: The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87-99%); 61/64), and specificity of 100% (95% CI 86-100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29-96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105). CONCLUSION: This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings

Molecular markers of anti-malarial drug resistance in Central, West and East African children with severe malaria.

BACKGROUND: The Plasmodium falciparum multidrug resistance 1 (PfMDR1), P. falciparum Ca(2+)-ATPase (PfATP6) and Kelch-13 propeller domain (PfK13) loci are molecular markers of parasite susceptibility to anti-malarial drugs. Their frequency distributions were determined in the isolates collected from children with severe malaria originating from three African countries. METHODS: Samples from 287 children with severe malaria [(Gabon: n = 114); (Ghana: n = 89); (Kenya: n = 84)] were genotyped for pfmdr1, pfatp6 and pfk13 loci by DNA sequencing and assessing pfmdr1 copy number variation (CNV) by real-time PCR. RESULTS: Pfmdr1-N86Y mutation was detected in 48, 10 and 10% in Lambaréné, Kumasi and Kisumu, respectively. At codon 184, the prevalence of the mutation was 73% in Lambaréné, 63% in Kumasi and 49% Kisumu. The S1034C and N1042D variants were absent at all three sites, while the frequency of the D1246Y mutation was 1, 3 and 13% in Lambaréné, Kumasi and Kisumu, respectively. Isolates with two pfmdr1 gene copy number predominantly harboured the N86Y wild-type allele and were mostly found in Kumasi (10%) (P < 0.0001). Among the main pfmdr1 haplotypes (NFD, NYD and YFD), NYD was associated with highest parasitaemia (P = 0.04). At the pfatp6 locus, H243Y and A623E mutations were observed at very low frequency at all three sites. The prevalence of the pfatp6 E431K variant was 6, 18 and 17% in Lambaréné, Kumasi and Kisumu, respectively. The L263E and S769N mutations were absent in all isolates. The pfk13 variants associated with artemisinin resistance in Southeast Asia were not observed. Eleven novel substitutions in the pfk13 locus occurring at low frequency were observed. CONCLUSIONS: Artemisinins are still highly efficacious in large malaria-endemic regions though declining efficacy has occurred in Southeast Asia. The return of chloroquine-sensitive strains following the removal of drug pressure is observed. However, selection of wild-type alleles in the multidrug-resistance gene and the increased gene copy number is associated with reduced lumefantrine sensitivity. This study indicates a need to constantly monitor drug resistance to artemisinin in field isolates from malaria-endemic countries

Malaria vector research and control in Haiti: a systematic review

BACKGROUND: Haiti has a set a target of eliminating malaria by 2020. However, information on malaria vector research in Haiti is not well known. This paper presents results from a systematic review of the literature on malaria vector research, bionomics and control in Haiti. METHODS: A systematic search of literature published in French, Spanish and English languages was conducted in 2015 using Pubmed (MEDLINE), Google Scholar, EMBASE, JSTOR WHOLIS and Web of Science databases as well other grey literature sources such as USAID, and PAHO. The following search terms were used: malaria, Haiti, Anopheles, and vector control. RESULTS: A total of 132 references were identified with 40 high quality references deemed relevant and included in this review. Six references dealt with mosquito distribution, seven with larval mosquito ecology, 16 with adult mosquito ecology, three with entomological indicators of malaria transmission, eight with insecticide resistance, one with sero-epidemiology and 16 with vector control. In the last 15 years (2000–2015), there have only been four published papers and three-scientific meeting abstracts on entomology for malaria in Haiti. Overall, the general literature on malaria vector research in Haiti is limited and dated. DISCUSSION: Entomological information generated from past studies in Haiti will contribute to the development of strategies to achieve malaria elimination on Hispaniola. However it is of paramount importance that malaria vector research in Haiti is updated to inform decision-making for vector control strategies in support of malaria elimination