ah Amir

Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi.

Significance Plasmodium knowlesi is a parasite that naturally infects cynomolgus monkeys but is also a major cause of severe zoonotic malaria in humans in South East Asia. Comparing the genomes of parasites restricted to growth in culture with cynomolgus RBCs and those adapted to growth in human RBCs identified a gene specifically required for invasion of human RBCs, a process that is critical for parasite replication. This gene encodes normocyte-binding protein Xa, a protein previously shown to bind human RBCs and implicated in invasion. Disruption of this gene blocks invasion of human but not cynomolgus RBCs, thus confirming a key mediator of human infection and a potential target for inclusion in vaccines to prevent human infection. The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

Invasion characteristics of a Plasmodium knowlesi line newly isolated from a human

Plasmodium knowlesi is extensively used as an important malaria model and is now recognized as an important cause of human malaria in Malaysia. The strains of P. knowlesi currently used for research were isolated many decades ago, raising concerns that they might no longer be representative of contemporary parasite populations. We derived a new P. knowlesi line (University Malaya line, UM01), from a patient admitted in Kuala Lumpur, Malaysia, and compared it with a human-adapted laboratory line (A1-H.1) derived from the P. knowlesi H strain. The UM01 and A1-H.1 lines readily invade human and macaque (Macaca fascicularis) normocytes with a preference for reticulocytes. Whereas invasion of human red blood cells was dependent on the presence of the Duffy antigen/receptor for chemokines (DARC) for both parasite lines, this was not the case for macaque red blood cells. Nonetheless, differences in invasion efficiency, gametocyte production and the length of the asexual cycle were noted between the two lines. It would be judicious to isolate and characterise numerous P. knowlesi lines for use in future experimental investigations of this zoonotic species.

Phylogenetic study of six species of Anopheles mosquitoes in Peninsular Malaysia based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA

BackgroundMolecular techniques are invaluable for investigation on the biodiversity of Anopheles mosquitoes. This study aimed at investigating the spatial-genetic variations among Anopheles mosquitoes from different areas of Peninsular Malaysia, as well as deciphering evolutionary relationships of the local Anopheles mosquitoes with the mosquitoes from neighbouring countries using the anopheline ITS2 rDNA gene.MethodsMosquitoes were collected, identified, dissected to check infection status, and DNA extraction was performed for PCR with primers targeting the ITS2 rDNA region. Sequencing was done and phylogenetic tree was constructed to study the evolutionary relationship among Anopheles mosquitoes within Peninsular Malaysia, as well as across the Asian region.ResultsA total of 133 Anopheles mosquitoes consisting of six different species were collected from eight different locations across Peninsular Malaysia. Of these, 65 ITS2 rDNA sequences were obtained. The ITS2 rDNA amplicons of the studied species were of different sizes. One collected species, Anopheles sinensis, shows two distinct pools of population in Peninsular Malaysia, suggesting evolvement of geographic race or allopatric speciation.ConclusionAnopheles mosquitoes from Peninsular Malaysia show close evolutionary relationship with the Asian anophelines. Nevertheless, genetic differences due to geographical segregation can be seen. Meanwhile, some Anopheles mosquitoes in Peninsular Malaysia show vicariance, exemplified by the emergence of distinct cluster of An. sinensis population.

Colonization of Anopheles cracens: a malaria vector of emerging importance

BackgroundAnopheles cracens has been incriminated as a vector for the simian malaria parasite, Plasmodium knowlesi, that is the fifth Plasmodium species infecting humans. Little experimental data exists on this mosquito species due to the lack of its availability in laboratories.FindingsThe population of An. cracens, collected from Kuala Lipis, Pahang was maintained at the insectary of the Department of Parasitology, Faculty of Medicine, University Malaya at 24-26°C and 60-80% relative humidity. The mosquitoes were maintained with artificial mating and blood-fed on humans and hamsters. The colony has been established since November 2011 and to date has reached its sixth generation.ConclusionThis is the first description of maintaining the Malaysian strain An. cracens colony by artificial mating. Colonization of An. cracens will provide fundamental information for genetic studies and will be useful in assessing comparative susceptibility to Plasmodium parasites.

Reduced red blood cell deformability in Plasmodium knowlesi malaria

The simian parasite Plasmodium knowlesi can cause severe and fatal human malaria. However, little is known about the pathogenesis of this disease. In falciparum malaria, reduced red blood cell deformability (RBC-D) contributes to microvascular obstruction and impaired organ perfusion. In P knowlesi infection, impaired microcirculatory flow has been observed in Macaca mulatta (rhesus macaques), unnatural hosts who develop severe and fatal disease. However, RBC-D has not been measured in human infection or in the natural host M fascicularis (long-tailed macaques). Using ektacytometry, we measured RBC-D in adults with severe and non-severe knowlesi and falciparum malaria and in healthy controls. In addition, we used micropipette aspiration to determine the relative stiffness of infected RBCs (iRBCs) and uninfected RBCs (uRBCs) in P knowlesi-infected humans and M fascicularis Ektacytometry demonstrated that RBC-D overall was reduced in human knowlesi malaria in proportion to disease severity, and in severe knowlesi malaria, it was comparable to that of severe falciparum malaria. RBC-D correlated inversely with parasitemia and lactate in knowlesi malaria and HRP2 in falciparum malaria, and it correlated with hemoglobin nadir in knowlesi malaria. Micropipette aspiration confirmed that in humans, P knowlesi infection increased stiffness of both iRBCs and uRBCs, with the latter mostly the result of echinocytosis. In contrast, in the natural host M fascicularis, echinocyte formation was not observed, and the RBC-D of uRBCs was unaffected. In unnatural primate hosts of P knowlesi, including humans, reduced deformability of iRBCs and uRBCs may represent a key pathogenic mechanism leading to microvascular accumulation, impaired organ perfusion, and anemia.

Autoantibody profile of patients infected with knowlesi malaria.

BACKGROUND Autoantibodies or antibodies against self-antigens are produced either during physiological processes to maintain homeostasis or pathological process such as trauma and infection. Infection with parasites including Plasmodium has been shown to generally induce elevated self-antibody (autoantibody) levels. Plasmodium knowlesi is increasingly recognized as one of the most important emerging human malaria in Southeast Asia that can cause severe infection leading to mortality. Autoimmune-like phenomena have been hypothesized to play a role in the protective immune responses in malaria infection. METHODS We studied the autoantibody profile from serum of eleven patients diagnosed with P. knowlesi. Autoantigen arrays were used to elucidate the autoantibody repertoire of P. knowlesi infected patients. The patented OGT Discovery Array with 1636 correctly folded antigen was employed. RESULTS Analysis of the patient versus control sera gave us 24 antigens with high reactivity with serum antibodies. CONCLUSIONS Understanding the autoantibody profile of malarious patients infected with P. knowlesi would help to further understand the host-parasite interaction, host immune response and disease pathogenesis. These reactive antigens may serve as potential biomarkers for cases of asymptomatic malaria and mild malaria or predictive markers for severe malaria.

Enterobius vermicularis Salpingitis Seen in the Setting of Ectopic Pregnancy in a Malaysian Patient

ABSTRACT We report a rare and unusual case of invasive Enterobius vermicularis infection in a fallopian tube. The patient was a 23-year-old Malaysian woman who presented with suprapubic pain and vaginal bleeding. A clinical diagnosis of ruptured right ovarian ectopic pregnancy was made. She underwent a laparotomy with a right salpingo-oophorectomy. Histopathological examination of the right fallopian tube showed eggs and adult remnants of E. vermicularis, and the results were confirmed using PCR and DNA sequencing.

Anisakiasis Causing Acute Dysentery in Malaysia.

Human anisakiasis is a zoonosis acquired by eating raw or undercooked infected seafood. Herein, we report a case of acute dysentery caused by anisakiasis in a 64-year-old man in Malaysia. A colonoscopy was performed and a nematode larva was found penetrating the mucosa of the ascending colon. Bleeding was observed at the site of penetration. Y-shaped lateral epidermal cords were seen from the cross section of the worm, which is a prominent feature of Anisakis larva. Molecular analysis using polymerase chain reaction of cytochrome oxidase 2 (cox2) gene confirmed the specimen to be larva of Anisakis simplex.

Fatal case of amoebic liver abscess in a child.

We reported a case of amoebic liver abscess (ALA) in a 6-year-old Malaysian boy who presented with fever, lethargy, diarrhoea and right hypochondriac pain. On admission he was diagnosed with perforated acute appendicitis and a laparotomy was done. After surgery he developed acute respiratory distress. Ultrasonography, chest X-Ray and CT scan revealed two ALAs in the posterior segment of right lobe of liver, pleural effusion and collapsed consolidation of lungs bilaterally. Percutaneous liver abscesses drainage was done and intravenous Metronidazole was started. PCR carried out on the pus from the abscess was positive for Entamoeba histolytica. Patient however succumbed to the infection one week after admission.

Diagnostic tools in childhood malaria

Every year, millions of people are burdened with malaria. An estimated 429,000 casualties were reported in 2015, with the majority made up of children under five years old. Early and accurate diagnosis of malaria is of paramount importance to ensure appropriate administration of treatment. This minimizes the risk of parasite resistance development, reduces drug wastage and unnecessary adverse reaction to antimalarial drugs. Malaria diagnostic tools have expanded beyond the conventional microscopic examination of Giemsa-stained blood films. Contemporary and innovative techniques have emerged, mainly the rapid diagnostic tests (RDT) and other molecular diagnostic methods such as PCR, qPCR and loop-mediated isothermal amplification (LAMP). Even microscopic diagnosis has gone through a paradigm shift with the development of new techniques such as the quantitative buffy coat (QBC) method and the Partec rapid malaria test. This review explores the different diagnostic tools available for childhood malaria, each with their characteristic strengths and limitations. These tools play an important role in making an accurate malaria diagnosis to ensure that the use of anti-malaria are rationalized and that presumptive diagnosis would only be a thing of the past.