Population genomic structure and adaptation in the zoonotic malaria parasite Plasmodium knowlesi

Malaria cases caused by the zoonotic parasite Plasmodium knowlesi are being increasingly reported throughout Southeast Asia and in travelers returning from the region. To test for evidence of signatures of selection or unusual population structure in this parasite, we surveyed genome sequence diversity in 48 clinical isolates recently sampled from Malaysian Borneo and in five lines maintained in laboratory rhesus macaques after isolation in the 1960s from Peninsular Malaysia and the Philippines. Overall genomewide nucleotide diversity (π = 6.03 × 10⁻³) was much higher than has been seen in worldwide samples of either of the major endemic malaria parasite species Plasmodium falciparum and Plasmodium vivax. A remarkable substructure is revealed within P. knowlesi, consisting of two major sympatric clusters of the clinical isolates and a third cluster comprising the laboratory isolates. There was deep differentiation between the two clusters of clinical isolates [mean genomewide fixation index (F_ST) = 0.21, with 9,293 SNPs having fixed differences of F_ST = 1.0]. This differentiation showed marked heterogeneity across the genome, with mean F_ST values of different chromosomes ranging from 0.08 to 0.34 and with further significant variation across regions within several chromosomes. Analysis of the largest cluster (cluster 1, 38 isolates) indicated long-term population growth, with negatively skewed allele frequency distributions (genomewide average Tajima’s D = −1.35). Against this background there was evidence of balancing selection on particular genes, including the circumsporozoite protein (csp) gene, which had the top Tajima’s D value (1.57), and scans of haplotype homozygosity implicate several genomic regions as being under recent positive selection.The zoonotic malaria parasite Plasmodium knowlesi is a significant cause of human malaria, with a wide spectrum of clinical outcomes including high parasitemia and death (1–4). Long known as a malaria parasite of long-tailed and pig-tailed macaques (5), the first large focus of human cases was described only in 2004 in the Kapit Division of Sarawak in Malaysian Borneo (6). Since then infections have been described from almost all countries in Southeast Asia (2, 7). Travelers to the region from Europe, North America, and Australasia also have recently acquired P. knowlesi malaria (7, 8). Until the application of molecular assays for specific detection, human P. knowlesi malaria was largely misdiagnosed as Plasmodium malariae, a morphologically similar but distantly related species (1, 6, 9, 10). Studies in the Kapit Division of Sarawak in Malaysian Borneo have indicated that P. knowlesi malaria is primarily a zoonosis with macaques as reservoir hosts (11) and that the forest-dwelling mosquito species Anopheles latens is the local vector for P. knowlesi (12). Other members of the Anopheles leucosphyrus group are vectors in different parts of Southeast Asia and may determine the geographical distribution of transmission (13, 14).Although P. knowlesi malaria is regarded as an emerging infection, there clearly have been increased efforts in detection made since its existence as a significant zoonosis was discovered, and specific detection has been enhanced by the declining numbers of human cases caused by other malaria parasites in Southeast Asia (15). Aside from the first two human cases described several decades ago (5), there is direct evidence of human P. knowlesi infections from ∼20 y ago in Malaysian Borneo and Thailand obtained by retrospective molecular analysis of material from archived blood spots and slides (10, 16), and molecular population genetic evidence indicates the zoonosis has been in existence for a much longer time (11). The genetic diversity of P. knowlesi is high within humans as well as macaques, with sequence data on three loci [the circumsporozoite protein (csp) gene, 18S rRNA, and mtDNA genome] indicating extensive shared polymorphism and no fixed differences between P. knowlesi parasites from humans and monkeys sampled in the same area in Sarawak, Malaysian Borneo (6, 11). Analysis of samples from a smaller number of humans and monkeys in Thailand showed alleles of the P. knowlesi merozoite surface protein 1 (msp1) gene to be similarly diverse in both hosts (16), and there were shared polymorphisms of the csp gene in parasites from a few infections examined in humans and macaques in Singapore (17). Recent multilocus microsatellite analysis has indicated a deep population subdivision in P. knowlesi associated with long-tailed and pig-tailed macaques; both major types infect humans and occur sympatrically at most sites in Malaysia, but the two types show some additional geographical differentiation across sites (18).Human populations have grown very rapidly in the Southeast Asian region and encroach on most of the wild macaque habitats, so it is vital to know if P. knowlesi parasites are adapting to human hosts or to anthropophilic mosquito vector species, either of which could cause human–mosquito–human transmission. Initial analysis of the P. knowlesi reference genome sequence (strain H) highlighted some unique features of the genome of this species (19), namely, schizont infected cell agglutination variant (SICAvar) and knowlesi interspersed repeat (KIR) variant antigen genes, which were widely dispersed instead of being predominantly localized in subtelomeric regions as seen in large gene families in Plasmodium falciparum and Plasmodium vivax (20). Here, we analyzed genomewide diversity in P. knowlesi and conducted scans for signatures of balancing and directional selection, revealing extremely high genetic diversity and significant structuring of this species into subpopulation clusters that appear to be reproductively isolated as well as loci that show evidence of recent strong selection.     

Adrenocorticotropin and adenosine 3',5'-monophosphate stimulate de novo synthesis of adrenal phosphatidic acid by a cycloheximide-sensitive, CA++-dependent mechanism

We tested further our postulate that enhanced de novo synthesis of phosphatidic acid is responsible for ACTH- and cAMP-induced increases in adrenal phospholipids in the phosphatidate polyphosphoinositide pathway. During incubation of adrenal sections or cells in vitro, ACTH and cAMP increased the concentrations of and incorporation of [3H]glycerol and [14C]palmitate into phosphatidylcholine and phosphatidylethanolamine, two major phospholipids which are derived from phosphatidic acid, but are extrinsic to the inositide pathway. Thus, it is unlikely that ACTH and cAMP increase inositide phospholipids at the expense of other phospholipids. Similar to previously reported effects on phosphatidic acid and inositide phospholipids, cycloheximide blocked the effects of ACTH and cAMP on phosphatidylcholine and phosphatidylethanolamine. In addition, Ca++ was required for these effects, as well as for cAMP-induced increases in phosphatidic acid, inositide phospholipids, and steroidogenesis. Our findings strongly suggest that ACTH, via cAMP, stimulates de novo phosphatidate synthesis by a cycloheximide-sensitive, Ca++-dependent process, and this stimulation causes a rapid generalized increase in adrenal phospholipids. Moreover, the increased incorporation of labeled glycerol and palmitate into phospholipids suggests that ACTH and cAMP may stimulate the glycerol-3'-PO4 acyltransferase reaction. This stimulatory effect may play a central role in the steroidogenic and trophic actions of ACTH and cAMP.     

Non-dysraphic intramedullary spinal cord lipoma

True intramedullary spinal cord lipomas are extremely rare. Two cases of intramedullary spinal cord lipoma are presented. The patients did not exhibit any form of spinal dysraphism. The patients presented with gait difficulty, upper limb weakness, sphincter disturbance, dysesthesias and neck pain. The tumors were removed sub-totally and the neurological grade improved postoperatively in one of the patients.     

A national survey of obstetric anaesthetic handovers

The handover of patient information between shifts enables continuity of care and increases patient safety. We surveyed UK practice during handovers in obstetric anaesthesia. A questionnaire was sent to 239 lead consultant obstetric anaesthetists to record routine practice in their unit and individual opinion about handover procedures. Responses were received from 168 anaesthetists, a 70% response rate. Handover policies were available in 10% of units. Most (76%) responding units had an allocated time for handover. In most units (76%), the duration of handover was reported as being < 15 min but the actual duration and depth of any discussion involved were not specified. Handovers were rarely documented in writing (7%). Consultant anaesthetists were most likely to be present at the morning handover and few handovers were multidisciplinary. Four percent of units reported critical incidents following inadequate handovers in the past 12 months. We identify features in handover procedures that could be improved.     

The mechanism of action of insulin on phospholipid metabolism in rat adipose tissue. Requirement for protein synthesis and a carbohydrate source, and relationship to activation of pyruvate dehydrogenase

We studied certain metabolic requirements for insulin-induced increases in phospholipids, and the relationship of phospholipid changes to the insulin-induced activation of pyruvate dehydrogenase, in rat adipocytes and fat pads in vitro. Increases in the contents of phosphatidylinositol and phosphatidylserine mass were maximal in rat fat pads within 10 min of incubation with insulin, and preceded or accompanied measurable increases in pyruvate dehydrogenase activity. In dose-response studies, the contents of these phospholipids and pyruvate dehydrogenase activity increased in parallel in response to increasing concentrations of insulin. Cycloheximide and puromycin inhibited insulin-induced increases in the mass of both of these phospholipids, as well as (in confirmation of previous reports) pyruvate dehydrogenase activity. Effects of insulin on phospholipid metabolism and pyruvate dehydrogenase were found to require an exogenous carbohydrate source, and fructose was nearly as effective as glucose in this regard. Insulin-induced increases in phosphatidylinositol and phosphatidylserine were demonstrated in the mitochondrial fraction, which is also the subcellular locus of pyruvate dehydrogenase. The present findings suggest that there is a relationship between insulin-induced increases in phospholipids and pyruvate dehydrogenase activity, but the nature of this relationship remains to be defined.     

The changing spectrum of severe falciparum malaria: a clinical study from Bikaner (northwest India)

BACKGROUND & OBJECTIVES: Recently there were reports from all over India about changing spectrum of clinical presentation of severe malaria. The present study was planned to study the same in the northwest India. METHODS: This prospective study was conducted on patients of severe malaria admitted in a classified malaria ward of a tertiary care hospital in Bikaner, Rajasthan (northwest India) during 1994 and 2001. It included adult patients of both sexes belonging to all age groups. The diagnosis of Plasmodium falciparum was confirmed by demonstrating asexual form of parasites in peripheral blood smear. All patients were treated with i.v./oral quinine. The specific complications were treated by standard WHO protocol. The data for individual complications for both the years were analysed by applying chi-square test. RESULTS: In a prospective study in 1994 the spectrum of complication was dominated by cerebral malaria (25.75%) followed by jaundice (11.47%), bleeding tendencies (9.59%), severe anaemia (5.83%), shock (5.26%), Acute respiratory distress syndrome-ARDS (3.01%), renal failure (2.07%) and hypoglycemia (2.07%) whereas in 2001 it was dominated by jaundice (58.85%) followed by severe anaemia (26.04%), bleeding tendencies (25.52%), shock (10.94%), cerebral malaria (10.94%), renal failure (6.25%), ARDS (2.08%) and hypoglycemia (1.56%). The sharp difference for presence of jaundice and severe anaemia in 2001 and cerebral malaria in 1994 was statistically significant. Similarly, the important cause of mortality in 2001 was multiple organ dysfunction syndrome (71.10%) with predominant presentation of jaundice and renal failure, whereas in 1994, it was cerebral malaria (77.96%). INTERPRETATION & CONCLUSION: The observation of changing spectrum of severe malaria in this study and a significant increase in presentation with jaundice as an important manifestation is highly essential for primary, secondary and tertiary level health care providers for proper diagnosis and management.