Natural Human Infections with Plasmodium cynomolgi, P. inui, and 4 other Simian Malaria Parasites, Malaysia

We detected the simian malaria parasites Plasmodium knowlesi, P. cynomolgi, P. inui, P. coatneyi, P. inui–like, and P. simiovale among forest fringe–living indigenous communities from various locations in Malaysia. Our findings underscore the importance of using molecular tools to identify newly emergent malaria parasites in humans

Plasmodium knowlesi malaria an emerging public health problem in Hulu Selangor, Selangor, Malaysia (2009–2013) : epidemiologic and entomologic analysis.

Background: While transmission of the human Plasmodium species has declined, a significant increase in Plasmodium knowlesi/Plasmodium malariae cases was reported in Hulu Selangor, Selangor, Malaysia. Thus, a study was undertaken to determine the epidemiology and the vectors involved in the transmission of knowlesi malaria. Methods: Cases of knowlesi/malariae malaria in the Hulu Selangor district were retrospectively reviewed and analyzed from 2009 to 2013. Mosquitoes were collected from areas where cases occurred in order to determine the vectors. Leucosphyrus group of mosquitoes were genetically characterized targeting the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit I (CO1). In addition, temporal and spatial analyses were carried out for human cases and vectors. Results: Of the 100 microscopy diagnosed P. knowlesi/P. malariae cases over the 5 year period in the Hulu Selangor district, there was predominance of P. knowlesi/P. malariae cases among the young adults (ages 20–39 years; 67 cases; 67%). The majority of the infected people were involved in occupations related to agriculture and forestry (51; 51%). No death was recorded in all these cases. Five hundred and thirty five mosquitoes belonging to 14 species were obtained during the study. Anopheles maculatus was the predominant species (49.5%) followed by Anopheles letifer (13.1%) and Anopheles introlatus (11.6%). Molecular and phylogenetic analysis confirmed the species of the Leucosphyrus group to be An. introlatus. In the present study, only An. introlatus was positive for oocysts. Kernel Density analysis showed that P. knowlesi hotspot areas overlapped with areas where the infected An. introlatus was discovered. This further strengthens the hypothesis that An. introlatusis is the vector for P. knowlesi in the Hulu Selangor district. Unless more information is obtained on the vectors as well as macaque involved in the transmission, it will be difficult to plan effective control strategies. The utilization of modern analytical tools such as GIS (Geographic Information System) is crucial in estimating hotspot areas for targeted control strategies. Conclusions: Anopheles introlatus has been incriminated as vector of P. knowlesi in Hulu Selangor. The cases of P. knowlesi are on the increase and further research using molecular techniques is needed

Pathogenesis of Plasmodium berghei ANKA infection in the gerbil (Meriones unguiculatus) as an experimental model for severe malaria

Background: As the quest to eradicate malaria continues, there remains a need to gain further understanding of the disease, particularly with regard to pathogenesis. This is facilitated, apart from in vitro and clinical studies, mainly via in vivo mouse model studies. However, there are few studies that have used gerbils (Meriones unguiculatus) as animal models. Thus, this study is aimed at characterizing the effects of Plasmodium berghei ANKA (PbA) infection in gerbils, as well as the underlying pathogenesis. Methods: Gerbils, 5-7 weeks old were infected by PbA via intraperitoneal injection of 1 × 106 (0.2 mL) infected red blood cells. Parasitemia, weight gain/loss, hemoglobin concentration, red blood cell count and body temperature changes in both control and infected groups were monitored over a duration of 13 days. RNA was extracted from the brain, spleen and whole blood to assess the immune response to PbA infection. Organs including the brain, spleen, heart, liver, kidneys and lungs were removed aseptically for histopathology. Results: Gerbils were susceptible to PbA infection, showing significant decreases in the hemoglobin concentration, RBC counts, body weights and body temperature, over the course of the infection. There were no neurological signs observed. Both pro-inflammatory (IFNγ and TNF) and anti-inflammatory (IL-10) cytokines were significantly elevated. Splenomegaly and hepatomegaly were also observed. PbA parasitized RBCs were observed in the organs, using routine light microscopy and in situ hybridization. Conclusion: Gerbils may serve as a good model for severe malaria to further understand its pathogenesis

Pathogenesis of

Background: As the quest to eradicate malaria continues, there remains a need to gain further understanding of the disease, particularly with regard to pathogenesis. This is facilitated, apart from in vitro and clinical studies, mainly via in vivo mouse model studies. However, there are few studies that have used gerbils (Meriones unguiculatus) as animal models. Thus, this study is aimed at characterizing the effects of Plasmodium berghei ANKA (PbA) infection in gerbils, as well as the underlying pathogenesis. Methods: Gerbils, 5-7 weeks old were infected by PbA via intraperitoneal injection of 1 × 106 (0.2 mL) infected red blood cells. Parasitemia, weight gain/loss, hemoglobin concentration, red blood cell count and body temperature changes in both control and infected groups were monitored over a duration of 13 days. RNA was extracted from the brain, spleen and whole blood to assess the immune response to PbA infection. Organs including the brain, spleen, heart, liver, kidneys and lungs were removed aseptically for histopathology. Results: Gerbils were susceptible to PbA infection, showing significant decreases in the hemoglobin concentration, RBC counts, body weights and body temperature, over the course of the infection. There were no neurological signs observed. Both pro-inflammatory (IFNγ and TNF) and anti-inflammatory (IL-10) cytokines were significantly elevated. Splenomegaly and hepatomegaly were also observed. PbA parasitized RBCs were observed in the organs, using routine light microscopy and in situ hybridization. Conclusion: Gerbils may serve as a good model for severe malaria to further understand its pathogenesis

Ratio of IL-10:IFN-γ, IL-10:TNF and IL-10:CXCL10 protein in the presence and absence of HBEC.

<p>Columns and vertical bars represent means ± SEM of three experiments. Statistical significance (***p<0.001) was assessed using ANOVA and the Bonferroni post hoc test.</p

Effect of caspase-1 inhibition on IFN-γ and IL-1β production in co-cultures of PBMC and iRBC.

<p>YVAD did not affect IFN-γ production but did inhibit that of IL-1β. uRBC = unparasitised red blood cells. Control  =  no PBMC, uRBC or iRBC. YVAD (50 µmol/L), a caspase-1 inhibitor, was pre-incubated with PBMC and HBEC for 30 min prior to addition of the iRBCs. At 24 h, supernates were analysed for IFN-γ and IL-1β. Columns represent values from a single experiment.</p

Effects of MHC II, ICOSL, IL-2 neutralisation on IFN-γ production in the tripartite cultures.

<p>Individually, anti-MHC II (A), anti-ICOSL (B), anti-IL-2 (C) and Orencia (D) did not affect IFN-γ production. However, when all treatments were combined they did reduce the endothelial cell-mediated enhancement of IFN-γ (E). uRBC  =  unparasitised red blood cells. Control  =  no PBMC, uRBC or iRBC. MHC II, ICOSL and IL-2 neutralising antibodies, and Orencia, were incubated with the tripartite cultures for 24 h. Supernates were then harvested and analysed for IFN-γ. Columns represent means of two separate experiments.</p

Effect of NK depletion on IFN-γ production in tripartite cultures.

<p>A. NK cell depletion strategy. B. IFN-γ protein production was decreased by NK cell depletion. uRBC  =  unparasitised red blood cells. Control  =  no PBMC, uRBC or iRBC. “PBMC N (NK depleted)” co-cultures contained PBMC that had been depleted of 99% NK cells stained with CD16, CD56 and NKp46 prior. “PBMC N” refers to co-cultures in which PBMC had not been depleted of NK cells. Co-cultures were 24 h, following which supernates were harvested and analysed for IFNγ. Columns represent means of two separate experiments.</p

Primer sequences used.

<p>Primer sequences used.</p