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.     

Normal pio-dural arterial connections

The arterial blood supply to the dura mater is rich, complex and is derived from both the internal and external carotid systems. Endovascular management of a variety of intracranial diseases necessitates a thorough understanding of the dural arterial network. In this article we review the normal contributions of the pial arteries to the blood supply of the dura mater and discuss some aspects of its role in the supply of dural arteriovenous shunts (DAVS).     

Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening.

BACKGROUND: Until recently, Plasmodium knowlesi malaria in humans was misdiagnosed as Plasmodium malariae malaria. The objectives of the present study were to determine the geographic distribution of P. knowlesi malaria in the human population in Malaysia and to investigate 4 suspected fatal cases. METHODS: Sensitive and specific nested polymerase chain reaction was used to identify all Plasmodium species present in (1) blood samples obtained from 960 patients with malaria who were hospitalized in Sarawak, Malaysian Borneo, during 2001-2006; (2) 54 P. malariae archival blood films from 15 districts in Sabah, Malaysian Borneo (during 2003-2005), and 4 districts in Pahang, Peninsular Malaysia (during 2004-2005); and (3) 4 patients whose suspected cause of death was P. knowlesi malaria. For the 4 latter cases, available clinical and laboratory data were reviewed. RESULTS: P. knowlesi DNA was detected in 266 (27.7%) of 960 of the samples from Sarawak hospitals, 41 (83.7%) of 49 from Sabah, and all 5 from Pahang. Only P. knowlesi DNA was detected in archival blood films from the 4 patients who died. All were hyperparasitemic and developed marked hepatorenal dysfunction. CONCLUSIONS: Human infection with P. knowlesi, commonly misidentified as the more benign P. malariae, are widely distributed across Malaysian Borneo and extend to Peninsular Malaysia. Because P. knowlesi replicates every 24 h, rapid diagnosis and prompt effective treatment are essential. In the absence of a specific routine diagnostic test for P. knowlesi malaria, we recommend that patients who reside in or have traveled to Southeast Asia and who have received a "P. malariae" hyperparasitemia diagnosis by microscopy receive intensive management as appropriate for severe falciparum malaria.     

Fibrillar IgA deposition may be associated with atypical dermatitis herpetiformis – a report of two cases

We report two cases of known dermatitis herpetiformis (DM) who presented with atypical clinical dermatological features. The first case, a 31-year-old woman, presented with an itchy urticated eruption on the abdomen and trunk, while the second case, a 50-year-old woman developed a scaly psoriasiform eruption on the extensor aspects of her knees and elbows. In each case, direct immunofluorescence of a perilesional skin biopsy showed a fibrillar pattern of IgA deposition at the basement membrane zone. Pre-embedding immunoelectron microscopy of these 2 specimens, using a 1 nm gold probe and silver enhancement, showed colloid gold panicle deposition around microfibril bundles in the dermal papillae. Recent studies have shown that fibrillin, a 350 kDa glycoprotein, is also associated with these microfibril bundles.
Serological tissue typing in our first case confirmed the presence of HLA-B8. B18. DR3 and DQw2 antigens consistent with dermatitis herpetiformis. In the second case, there was a notable absence of the antigens B17 and B27 typically found in psoriasis but HLA-AI, BX and DQ1 antigens were present, as commonly seen in dermatitis herpetiformis. Our findings would suggest that cases of dermatitis herpetiformis with fibrillar IgA deposition at the BMZ on direct IMF may show an atypical clinical presentation. In addition, our imunoelectron microscopy findings may provide a further clue to an ultrastructural abnormality in this disease.     

Purification and characterization of a putative fimbrial protein/receptor of Porphyromonas gingivalis

Porphyromonas gingivalis has been implicated as a major aetiological agent in certain forms of periodontal disease. P. gingivalis is a Gram-negative, asaccharolytic bacterium that obtains energy from the fermentation of amino acids derived from the hydrolysis of host protein. Virulence factors of this bacterium include the capsule, fimbrial adhesins, cytotoxins and extracellular hydrolytic enzymes. A 43 kDa fimbrillin from P. gingivalis has been isolated and characterized. However, there is evidence that a second type of fimbria exists on the surface of P. gingivalis. A putative P. gingivalis fimbrial protein from a membrane preparation has been isolated and identified. This protein was shown to be reactive with sera from patients harbouring P. gingivalis. A 28 kDa protein fragment was purified by anion exchange, gel filtration and reversed-phase chromatography. N-terminal sequence analysis of the 28 kDa protein fragment revealed homology to the fimbrial precursor protein of Dichelobacter nodosus. A peptide corresponding to the N-terminal 26 amino acyl residues of the 28 kDa protein fragment was synthesized and used to raise antibodies to the protein. Western blot analysis after SDS-PAGE of a P. gingivalis membrane preparation using the antibodies raised to the synthetic peptide detected three proteins of 36, 41 and 67 kDa. When protease inhibitors were not included in the extraction procedure only the 36 and 41 kDa bands were detected. It would appear, therefore, that the intact protein has an M_r of 67 kDa and that the 28, 36 and 41 kDa bands represent protein fragments produced by endogenous proteolytic activity. Based on sequence homology, the 67 kDa protein is possibly a sub-unit of a second P. gingivalis fimbrial type or a surface receptor.     

Molecular Mapping of the Major Epitopes of BP180 Recognized by Herpes Gestationis Autoantibodies

Herpes gestationis (HG) is an autoantibody-mediated subepidermal bullous dermatosis associated with pregnancy. The primary target of HG autoantibodies is BP180, a 180-kDa hemidesmosomal glycoprotein. We previously showed that autoantibodies and autoimmune T lymphocytes from HG patients recognize the MCW-1 antigenic site (AA 507-520), which is located in the membrane-proximal noncollagenous domain (NC16A) of BP180. Here, we analyzed the sera of 37 HG patients to further define the sites on BP180 that are targeted by autoantibodies. All of the HG sera, but none of the control sera, were immunoreactive with sec180e, a 120-kDa recombinant protein encompassing the entire BP180 extracellular domain. HG sera depleted of reactivity to NC16A no longer reacted with sec180e, indicating that the major HG-associated epitopes on BP180 are restricted to the NC16A domain. The vast majority of the HG sera (34 of 37) reacted with a 7 amino acid peptide corresponding to the N-terminal half of MCW-1 (MCW-1A). Eleven HG sera (including the 3 that failed to react with MCW-1A) recognized one or more of three antigenic sites located within a 15 amino acid stretch immediately downstream of MCW-1A. In summary, we have identified four major HG-associated epitopes clustered within a 22 amino acid region of the BP180 ectodomain. These findings support the hypothesis that an autoimmune response to the BP180 NC16A domain is a crucial step in the pathogenesis of HG.     

Peripelvic urinoma associated with benign pro static hypertrophy

A case report of a large peripelvic urinoma which disappeared after decompression of a distended obstructed bladder is presented. The etiology of this rare complication of bladder outlet obstruction is reviewed and discussed.     

Unusual variants of pemphigoid: from pruritus to pemphigoid nodularis

We report three patients with pemphigoid nodularis. Patients were females aged 76, 71 and 50 years, and all had features of bullous pemphigoid together with prurigo-like lesions at some stage of their illness. In two cases, nodular lesions preceded the onset of blistering by some months. Blisters arose on normal skin and in one patients also at sites of prurigo lesions. Routine histology of bullous lesions revealed the presence of subepidermal blisters. Electron microscopy performed in two cases confirmed the level of split to be through the lamina lucida. Direct immunofluorescence in all cases was positive, with linear basement membrane zone deposition of IgG and C3. Circulating IgG anti-basement membrane antibody was also detected in all patients, and in two, immunoblotting revealed a single antigen of 220 kD.