Staphylococcus aureus nasal carriage among healthcare workers in Kisangani, the Democratic Republic of the Congo.

Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, but there are few data from Central Africa. The objective of our study was to characterise S. aureus colonisation isolates from healthcare-exposed professionals in the Democratic Republic of the Congo (DRC). Healthcare workers and medical students (n = 380) in Kisangani, DRC were screened for S. aureus nasal carriage in a single-centre cross-sectional study in the University Hospital of Kisangani. The isolates were identified and characterised using phenotypic and genotypic methods. The nasal carriage rate of S. aureus was 16.6 % and 10 out of 63 isolates (15.9 %) were MRSA. We found 28 different spa types. Most MRSA isolates belonged to ST8-spa t1476-SCCmec V. The majority of MRSA were multidrug-resistant to non-beta-lactam antibiotics. Overall, 28.5 % of S. aureus carried Panton-Valentine leucocidin (PVL)-encoding genes (all methicillin-sensitive) and 17.5 % carried toxic shock syndrome toxin-1 (TSST-1)-encoding genes. The finding of MRSA carriage among healthcare workers in a setting with limited access to diagnostic microbiology and appropriate therapy calls for improved education on infection control practices and supports the introduction of surveillance programmes.

Pituitary adenylate cyclase-activating polypeptide deficiency associated with increased platelet count and aggregability in nephrotic syndrome.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently identified as an inhibitor of megakaryopoiesis and platelet aggregability. OBJECTIVE: We studied PACAP levels in children with nephrotic syndrome (NS), which is associated with thrombocytosis, platelet hyperaggregability, and an increased risk of thrombosis. PATIENTS/METHODS: In four children with congenital NS (CNS) and 24 children with idiopathic NS (INS), plasma and urine levels of PACAP and ceruloplasmin were measured, as were platelet counts and platelet aggregation responses to collagen. In CNS patients, in vitro megakaryopoiesis and nuclear factor-κB expression in platelet lysates were also measured. All tests were performed during the nephrotic state and the non-nephrotic state. RESULTS: Urinary losses of PACAP and ceruloplasmin were observed during the nephrotic state, and disappeared during the non-nephrotic state. Plasma PACAP deficiency was more pronounced in CNS patients than in INS patients. Thrombocytosis was observed in all CNS patients and in 11 of 29 INS patients during the nephrotic state. During the PACAP-deficient state, in vitro megakaryopoiesis was increased for CNS patients, and this effect could be reversed by the addition of recombinant PACAP. Platelet hyperaggregability was observed during the nephrotic state in both CNS and INS patients. In INS patients, the addition of recombinant PACAP to patients' platelets was studied, and resulted in decreased aggregation during the nephrotic state. Platelet aggregation correlated inversely with plasma PACAP levels, but not with serum albumin levels. CONCLUSIONS: We demonstrate urinary losses of PACAP and plasma PACAP deficiency in children with NS, associated with thrombocytosis and platelet hyperaggregability.

Update on the causes of platelet disorders and functional consequences.

Platelets are derived from megakaryocytes in the bone marrow that create the cellular machinery the platelet needs to participate in the different processes of primary hemostasis including adhesion, activation and clot formation at the site of injury. Defects related to megakaryocyte differentiation, platelet formation, and/or platelet function can result in bleeding. Patients with thrombopathies can present with mucous membrane bleeding but may also present with bleeding following trauma or surgery. In this review, we have classified inherited platelet bleeding disorders (IPD) according to their underlying defective pathway: transcription regulation, TPO signaling, cytoskeletal organization, apoptosis, granule trafficking, and receptor signaling. Platelet function testing has provided insights into the underlying molecular defects that can result in bleeding. A major step forward was made during the last 3 years using new-generation genetic approaches that resulted in the discovery of novel genes such as NBEAL2, RBM8A, ACTN1, and GFI1B for the well-known IPD that cause gray platelet syndrome, thrombocytopenia-absent radius syndrome, and autosomal dominant thrombocytopenias, respectively. In the near future, it is expected that a similar approach will identify many novel genes that cause IPD of unknown etiology, which are common. The future challenge will be to use a functional, systems biology approach to study the genes mutated in IPD and determine their roles in megakaryocyte and platelet biology and pathology.

Epidemic increase in Salmonella bloodstream infection in children, Bwamanda, the Democratic Republic of Congo.

Salmonella enterica is the leading cause of bloodstream infection in children in sub-Saharan Africa, but few data are available from Central-Africa. We documented during the period November 2011 to May 2012 an epidemic increase in invasive Salmonella bloodstream infections in HGR Bwamanda, a referral hospital in Equateur Province, DR Congo. Salmonella spp. represented 90.4 % (103 out of 114) of clinically significant blood culture isolates and comprised Salmonella Typhimurium (54.4 %, 56 out of 103), Salmonella Enteritidis (28.2 %, 29 out of 103) and Salmonella Typhi (17.5 %, 18 out of 103), with Salmonella Enteritidis accounting for most of the increase. Most (82 out of 103, 79.6 %) isolates were obtained from children < 5 years old. Median ages of patients infected with Salmonella Typhimurium and Salmonella Enteritidis were 14 months (14 days to 64 years) and 19 months (3 months to 8 years) respectively. Clinical presentation was non-specific; the in-hospital case fatality rate was 11.1 %. More than two thirds (69.7 %, 53 out of 76) of children < 5 years for whom laboratory data were available had Plasmodium falciparum infection. Most (83/85, 97.6 %) non-typhoid Salmonella isolates as well as 6/18 (33.3 %) Salmonella Typhi isolates were multidrug resistant (i.e. resistant to the first-line oral antibiotics amoxicillin, trimethoprim-sulfamethoxazole and chloramphenicol), one (1.0 %) Salmonella Typhimurium had decreased ciprofloxacin susceptibility owing to a point mutation in the gyrA gene (Gly81Cys). Multilocus variable-number tandem-repeat (MLVA) analysis of the Salmonella Enteritidis isolates revealed closely related patterns comprising three major and four minor profiles, with differences limited to one out of five loci. These data show an epidemic increase in clonally related multidrug-resistant Salmonella bloodstream infection in children in DR Congo.

Regulators of G protein signaling: role in hematopoiesis, megakaryopoiesis and platelet function.

Regulators of G protein signaling (RGS) are intracellular signaling regulators that bind activated G protein α subunits (Gα) and increase their intrinsic GTPase activity via their common RGS homology domain. In addition to their GTPase accelerating activity (GAP), RGS proteins also contain other domains that regulate their receptor selectivity, their interaction with other proteins such as adenylyl cyclase or their subcellular localization via interaction with scaffold proteins such as tubulin, 14-3-3 or spinophilin. There are at least 37 different RGS family members in humans and numerous physiological functions have been assigned to these proteins, which have rather a tissue-specific expression pattern. The role of some RGS proteins was shown to be important for hematopoiesis. More recent studies also focused on their expression in platelets, and for R4 RGS subfamily members RGS2, RGS16 and RGS18, it could be demonstrated that they regulate megakaryopoiesis and/or platelet function. These functional studies mostly comprised in vitro experiments and in vivo studies using small animal models. Their role in human pathology related to platelet dysfunction remains still largely unknown, except for a case report with a RGS2 gain of function mutation. In addition to an introduction on RGS signaling and different effectors with a special focus on the R4 subfamily members, we here will give an overview of the studies related to the role of RGS proteins in hematopoiesis, megakaryopoiesis and platelet function.

Proteomics to unravel platelet-related diseases and identify novel anti-platelet drugs.

Blood platelets play a fundamental role in primary haemostasis and wound repair, but are also involved in several thrombotic and bleeding disorders for which the underlying mechanisms are still largely unknown. Elucidating platelet biology would help in finding novel disease biomarkers and drug targets in complex and/or genetically unknown platelet-related disorders. Proteomics, which allows studying thousands of gene products at once, represents an efficient tool to quali-quantitatively analyze and compare the platelet protein patterns of different samples (i.e. control/patient, treated/untreated, drug sensitive/resistant), to investigate post-translation modifications, protein-protein interactions and the underlying molecular pathways. This review gives an overview of the applications of proteomic strategies to study platelet biology and function, as well as to unravel differences in protein expression according to specific platelet conditions (i.e. basic versus activated), compartments (i.e. membrane or granules) and fractions (i.e. phosphoproteins and glycoproteins). The use of innovative powerful proteomic technologies can lead to the identification of proteins whose expression is altered in pathological conditions, allowing the identification of candidate biomarkers for: i) understanding the molecular defects underlying platelet disorders, ii) obtaining novel insights in more complex diseases that involve platelets, iii) unraveling the drug mode of action or identifying the mechanisms of drug resistance and iv) detecting novel therapeutic antiplatelet targets based on fundamental platelet research studies. Several studies on how proteomics proved to be useful in our understanding of platelet function and its diseases are discussed. Eventually, this could result in the discovery of novel drug targets for antiplatelet therapy.

Recent advances in GNAS epigenetic research of pseudohypoparathyroidism.

Endocrinopathies in patients with hypocalcemia and hyperphosphatemia that share resistance to parathyroid hormone (PTH) are grouped under the term pseudohypoparathyroidism (PHP). Patients with PHP type Ia (PHP-Ia) often present with additional hormonal resistance and show characteristic physical features that are jointly termed as having an Albright's hereditary osteodystrophy (AHO) phenotype. Alternatively, PHPIb patients predominantly have PTH and sometimes TSH resistance but do not present with AHO features. Most of these PHP forms are caused by defects in GNAS, an imprinted gene locus consisting of maternal, paternal and biallelic transcripts. PHP-Ia is caused by heterozygous inactivating mutations in those exons of GNAS encoding the alpha subunit of the stimulatory guanine nucleotide-binding protein (Gsalpha) while PHPIb results from epigenetic GNAS defects. Familial and sporadic forms of PHP-Ib have distinct GNAS imprinting patterns: familial PHP-Ib patients have an exon A/B-only imprinting defect whereas sporadic PHP-Ib cases have abnormal imprinting of the three differentially methylated regions (DMRs) in GNAS. This classification of PHP was made years ago but was recently questioned since different studies showed GNAS epigenetic defects in PHP-Ia patients. In this review, we focus on the epigenetic description and screening methods of GNAS, the associated pathology and the recent need for a PHP reclassification.

Regulators of platelet cAMP levels: clinical and therapeutic implications.

Platelets are indispensable for primary haemostasis, but their function needs to be tightly regulated to prevent excessive platelet activity, possibly leading to atherothrombotic events. An important mediator of the platelet activity is cyclic AMP (cAMP), which inhibits platelet aggregation. Intracellular cAMP levels are regulated via the Gs and Gi alpha subunits of heterotrimeric G proteins, which couple to adenylyl cyclase to respectively stimulate or inhibit cAMP production. Binding of a ligand to its G protein-coupled seven-transmembrane receptor activates these G proteins. In this review, we discuss a Gs-coupled receptor on platelets, VPAC1, and 2 important Gi-coupled receptors, the ADP receptor P2Y(12) and the prostaglandin E(2) receptor EP3. The regulation of platelet cAMP levels at the level of the receptors themselves or the G proteins coupled to them is analyzed. Alterations in Gsα and Giα function are associated with altered platelet reactivity. An increase in Gs function, or alternatively a defective Gi signaling, can be a risk factor for bleeding, while a loss of Gs function can result in a prothrombotic state. Regulator of G protein signaling (RGS) proteins accelerate the rate of inactivation of G protein-mediated signaling. One of the RGS proteins, RGS2, inhibits Gs signaling by interacting directly with adenylyl cyclase. The thienopyridine class of antiplatelet agents is based on cAMP-mediated regulation of platelet function through modification of the P2Y(12) receptor. Clopidogrel and some other novel cAMP regulators are discussed. Secondly, we review the use of prostacyclin derivatives to treat pulmonary arterial hypertension.

Platelet Gs hypofunction and abnormal morphology resulting from a heterozygous RGS2 mutation.

SUMMARY BACKGROUND: Regulator of G-protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known. OBJECTIVE: To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology. METHODS: Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild-type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics. RESULTS: The mutation leads to reduced cAMP production after stimulation of Gs-coupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild-type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal alpha-granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change. CONCLUSIONS: We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.

ADP-degrading enzymes inhibit platelet activation in bile duct-ligated rats.

BACKGROUND: The effect of cholestatic liver disease on primary hemostasis function remains ill-defined. OBJECTIVES: To determine platelet function and identify the mechanisms involved in the observed platelet function in cholestatic rats. METHODS: Platelet function was studied in a model of 2-week bile duct ligation and compared to that in sham-operated rats with and without a storage pool defect. RESULTS: ADP-induced and collagen-induced platelet aggregation were clearly impaired following bile duct ligation (P<0.01 for areas under the curve). Crossover experiments, with sham platelets in bile duct-ligated plasma and vice versa, demonstrated that this is due to inhibition by a plasmatic factor, as sham platelets aggregated less in cholestatic plasma (P<0.03) and to an equal extent as platelets from bile duct-ligated rats when they were in the same sham or cholestatic plasma. Moreover, in bile duct-ligated rats, platelet ultrastructure was unaffected and platelet aggregation was similar to that of sham platelets when resuspended in the same plasma (P-value not significant). Additionally, studies in storage pool-deficient rats showed no role of platelet exhaustion. The plasmatic factor causing impaired aggregation was shown to be increased total activity of ADP-degrading enzymes upon bile duct ligation (P<0.01), as there was no decreased aggregation with a stable ADP analog in bile duct-ligated rats (P-value not significant vs. sham-operated rats). Furthermore, preincubation of plasma from bile duct-ligated rats with ADP decreased aggregation more than was seen with sham plasma (P<0.01). CONCLUSIONS: Bile duct ligation does not affect intrinsic platelet function, but impairs platelet activation via release of ADP-degrading enzymes in the circulation.

Polymorphisms and mutations of human TMPRSS6 in iron deficiency anemia.

Male subjects with iron deficiency from the general population were examined for polymorphisms or sporadic mutations in TMPRSS6 to identify genetic risk factors for iron deficiency anemia. Three uncommon non-synonymous polymorphisms were identified, G228D, R446W, and V795I (allele frequencies 0.0074, 0.023 and 0.0074 respectively), of which the R446W polymorphism appeared to be overrepresented in the anemic population. In addition, three children with iron refractory iron deficiency anemia, and one sibling with iron responsive iron deficiency anemia were also examined for polymorphisms or sporadic mutations in TMPRSS6. Two children (family 1) were compound heterozygotes for a L674F mutation and a previously described splicing defect predicted to cause skipping of exon 13 (IVS13+1 G>A). One child from the second family was homozygous for a deletion (497T) causing a frameshift (L166X+36) and premature termination. The sibling and mother from the second family were compound heterozygotes for the L166X mutation and the uncommon R446W polymorphism. Although in vitro expression studies demonstrated that the R446W isoform was biologically similar to wildtype Tmprss6, clinical data indicate that the R446W produces a milder disease when carried in trans with severe mutation in Tmprss6. The four children carrying mutations in TMPRSS6 all exhibited inappropriately high urinary hepcidin levels for the degree of iron deficiency.

Human platelet pathology related to defects in the G-protein signaling cascade.

Platelets are highly responsive to signals from their environment. The sensing and processing of some of these stimuli are mediated by G-protein signal transduction cascades. It is well established that proteins involved in signal transduction may be targets for naturally occurring mutations resulting in human diseases. The best-studied molecules in platelets in relation to disease are the G-protein coupled receptors being the most platelet-specific. Many of the other signal transduction genes are often not only present in platelets but also in other tissues. Therefore, the clinical phenotype of signaling defects in platelets, apart from the membrane receptor defects, is seldom isolated to a hemostatic phenotype. Moreover, as platelets are easily accessible cells, and one of the best-studied models regarding signaling, platelets are easily applicable to investigate defects in ubiquitously expressed genes. Apart from a discussion on classical thrombopathies, this review will also deal with the less commonly known relation between platelet signaling defects and disorders with a broader clinical phenotype.

A compound heterozygous mutation in glycoprotein VI in a patient with a bleeding disorder.

BACKGROUND: The physiological relevance of the collagen glycoprotein VI (GPVI) receptor was known prior to its recognition as a platelet membrane receptor as several patients lacking GPVI as a consequence of autoantibody inhibition presented with a mild bleeding diathesis. Remarkably, patients with a proven GPVI gene mutation have not yet been identified. RESULTS: In the present study, we describe a patient with a lifelong history of bleeding problems, structurally normal platelets but a functional platelet defect. Platelet aggregations are normal except for an absent response to Horm collagen, convulxin and the collagen-related peptide (CRP). ATP dense granule secretion is normal with ADP but absent with Horm collagen. Thrombus formation on a collagen surface in flowing blood is reduced but more single platelets are attached. Remarkably, the platelet function analyzer-100 shows a shortened collagen/ADP closure time. Flow cytometry demonstrates an absent expression of GPVI whereas immunoblot analysis shows strongly reduced levels of GPVI. The patient is compound heterozygous for an out-of-frame 16-bp deletion and a missense mutation S175N in a highly conserved residue of the 2nd Ig-like GPVI domain. The parents without clinical bleeding problems are heterozygous carriers. The mother carries the S175N mutation and presents with a mild functional platelet defect. In vitro studies show a reduced membrane expression and convulxin binding with the mutated S175N compared with the wild-type (WT) GPVI receptor. CONCLUSIONS: This study presents the first patient with a proven genetic GPVI defect.

The use of duplex doppler ultrasound in a case of multifocal hepatic hemangioma.

A one-month-old girl presented with multiple cutaneous hemangiomas, abnormal weight gain, hepatomegaly, and symptoms of bleeding disorder. Abdominal ultrasound and CT revealed a multifocal hypervascular hepatic tumor and signs of vascular overload. Biopsy confirmed the presence of an infantile hepatic hemangioma. Conservative treatment with high dose steroids showed regression of the hepatic lesions and the signs of vascular congestion. Ultrasound was used for follow-up and its role in treatment monitoring is discussed in this manuscript.

Review article: blood platelet number and function in chronic liver disease and cirrhosis.

BACKGROUND: The liver plays a central role in coagulation and fibrinolysis but is also closely intertwined with the function and number of blood platelets. AIM: To describe and integrate all literature concerning blood platelets and liver disease by performing a thorough literature research. METHODS: A thorough literature research on 'blood platelets' and 'liver disease' was performed. RESULTS: Thrombocytopenia is a marked feature of chronic liver disease and cirrhosis. Traditionally, this thrombocytopenia was attributed to passive platelet sequestration in the spleen. More recent insights suggest an increased platelet breakdown and to a lesser extent decreased platelet production plays a more important role. Besides the reduction in number, other studies suggest functional platelet defects. This platelet dysfunction is probably both intrinsic to the platelets and secondary to soluble plasma factors. It reflects not only a decrease in aggregability, but also an activation of the intrinsic inhibitory pathways. The net effect, finally, is a decreased platelet function in the various types of chronic liver diseases and cirrhosis. Finally, recent data suggest that platelets are not only affected by but can also contribute to the liver disease process, as for instance, in viral hepatitis and cholestatic liver disease. CONCLUSION: Platelet research in liver disease is a growing area of investigation and could provide new pathophysiological insights.

delta-Storage pool disease: a pitfall in the forensic investigation of sudden anal blood loss in children: a case report.

We present the case of a 3.5-year-old boy with sudden anal blood loss at school. Sexual abuse was suspected, and, apart from anal fissures seen on sigmoidoscopy, no other clinical signs of any sort of disorder were present. As no medical explanation for the blood loss could be given, penetrating anal trauma was suggested. During follow-up consultations, there were complaints of occasional blood loss. Platelet aggregation tests and electron microscopy finally helped diagnose a delta-storage pool disease which is a rare haemostatic disorder involving the dense granules of the platelets. Although exclusion of well-known blood diseases through routine laboratory testing is a common practice in children with sudden blood loss, this case illustrates the value of more specialised investigation both from a diagnostic and forensic point of view.

Video reveals self-stimulation in infancy.

UNLABELLED: The medical literature on early childhood masturbation is sparse. Only 12 patients who presented with infantile self-stimulation under the age of 1 y are described. During the last 2 y, five girls under 1 y of age presented at our department with self-stimulating behaviour. The diagnosis of this behaviour was difficult, but could be made by watching a video of the attacks. Infantile self-stimulation is often misdiagnosed and unnecessary investigations and useless treatments are often prescribed. Video recording can be of great help to put forward the correct diagnosis. Masturbation is not so uncommon and treatment consists mostly in reassuring the parents. It can, however, be associated with behavioural problems. Few data are available on the clinical outcome of childhood masturbation, but most children seem to develop normally. CONCLUSION: Infantile self-stimulation should always be considered in the differential diagnosis of "strange episodes or attacks".

Thrombopoiesis: new concepts.

Although platelets were already discovered light-microscopically in the 19th century, it became only clear in 1906 that they originate from megakaryocytes. The discovery of thrombopoietin in 1994 facilitated research on the origin of platelets, because this growth factor enabled expansion of megakaryocytes in culture. Thus, cell biological and molecular studies could be undertaken. Knowledge accumulated further by studying mouse models and by unravelling the defect in patients with hereditary thrombocytopenia. How a megakaryocyte originates from a hematopoietic stem cell and how this cell undergoes further maturation and differentiation is a complex process, controlled at different stages by several transcription factor. These transcription factors influence the expression of their own target genes, leading to megakaryocyte maturation and platelet release. One of the transcription factors that is most studied in this regard is GATA1, that forms a transcriptional complex with its cofactor FOG1. Families with hereditary thrombocytopenia have been described due to mutations in the GATA1 gene, and target genes were studied. This broadens our insight in normal megakaryocyte differentiation. The final release of platelets from the megakaryocyte is strongly dependent on the formation of so-called proplatelets. The formation of pseudopodia requires the presence of microtubuli. Beta 1-tubulin is a major part of these microtubuli and plays an important role not only in the genesis of platelets but also in the final discoid form of the platelet. Despite a renewed interest and expanding knowledge in this area, there are more questions than answers at this day.

A 16-year survey of Takayasu's arteritis in a tertiary Belgian center.

AIM: To evaluate the clinical features, angiographic findings and evolution of Takayasu's arteritis in a Belgian tertiary center, and to compare the findings with published series of Western patients. METHODS: Retrospective analysis of 15 patients with Takayasu's arteritis, satisfying the American College of Rheumathology criteria, in the period 1986 to 2002. Published series of Western patients were identified by means of a Medline search and citation-tracking. RESULTS: Diagnosis was often delayed, with a median period of 9 months. Patients presented with a variety of symptoms and clinical signs and had on average 4.5 arterial segments involved at angiography. Twelve patients received corticosteroid treatment and 4 of them additional immunosuppressive drugs. Five patients underwent angioplasty and/or stenting and 8 patients had open surgical procedures. During follow-up, there were 2 cardiac deaths and 2 other patients died from intracranial hemorrhage. Comparison with published series of other Western patients did not reveal major differences of anatomical distributions of the lesions, but left the impression that more aggressive use of immunosuppressive drugs might have improved the outcome. CONCLUSION: Takayasu's arteritis results in an important morbidity and mortality. More aggressive medical therapy may be advantageous, but this would require adequate investigation in a controlled trial for which a multicenter effort is needed because of the rarity of the disease.