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5 Chronic kidney disease mineral bone disorder in childrenLungu A.C., Constantinescu Iulia, Stoica Cristina

12 Diagnosis particularities in dystrophinopathies in womenPop Anita, Cornițescu M., Pleșca Doina Anca

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Abstract. Childhood and adolescence are critical times for the development of all organs and systems. Achievement of optimal bone mass in this period is thought to be the best predictor for bone health in the adult life1. In children, nutrition, physical activity, growth, endocrine and metabolic function is mandatory for a normal skeleton development and cardiovascular system. Disordered mineral and bone metabolism zassociated with chronic kidney disease causes important obstacles to final adult height, bone strength and cardiovascular integrity that may contribute to chronic morbidity[2, 3]. As kidney function decreases, a progressive deterioration in mineral homeostasis emerge, with an abnormal serum and tissue concentrations of phosphorus and calcium, and changes in circulating levels of hormones[4]. Therefore, treatment target of MBD includes maintaining optimal serum parameters for calcium, phosphorus and parathyroid hormone according to stage of CKD, in order to improve growth, high-turnover bone disease and prevent cardiovascular calcifications.Key words: chronic kidney disease, metabolic bone disorder, children

Lungu A.C.1, Constantinescu Iulia1, Stoica Cristina1

CHRONIC KIDNEY DISEASE MINERAL BONE DISORDER IN CHILDREN

REVIEWTherapeutics, Pharmacology and Clinical ToxicologyVol XIX, Number 1, March 2015Pages: 5 - 11© Copyright reserved 2015

Introduction

Childhood and adolescence are critical times for the development of a life-long bone health

and cardiovascular system. Bone growth involves a complex coordination of varied cell activities that are under the direct influence of a range of hormones and growth factors along with mineral metabolism that may be profoundly disturbed in chronic kidney disease[5]. This affects normal growth and favors cardiovascular calcifications that are indeed less prevalent than in adult population, but remains a leading cause of mortality[6]. The close connection between phosphocalcic metabolism disturbances, the treatment for correcting this alterations and vascular calcifications has led to a new and elaborate approach of chronic kidney disease in order to improve patients outcome. The term “renal osteodistrophy” fails to describe this complex association of physiopathological elements and the need to find an appropriate term was implied. Therefore, Kidney Disease: Improving Global Outcomes (KDIGO) ”established that that the term ‘CKD–Mineral and Bone Disorder (CKD–MBD)’ should be used to describe the broader clinical syndrome encompassing mineral, bone, and calcific cardiovascular abnormalities that develop as a complication of CKD”[4,7]. CKD-MBD is defined as being a systemic disorder of mineral and bone metabolism due to CKD manifested by either one or a combination of the following: (a) abnormalities of calcium, phosphorus, PTH, or vitamin D metabolism; (b) abnormalities in bone turnover, mineralization, volume, linear growth, or

(c) strength vascular or other soft-tissue calcification[4]. “Renal osteodistropy” on the other hand, describes only the bone impairment as a single complication of CKD that can be quantified by bone biopsy which describes three histologic parameters: three key histologic descriptors: bone turnover, mineralization, and volume (TMV) and has been recommended in all patients with CKD[3,4]. Although the histomorphometry of bone biopsy is the only available method for evaluate all three parameters, it is not routinely performed.

This review is intended to emphasize the current approach of mineral and bone disorder in children and the new therapeutically strategies, along with their impact on reducing mortality and improving long term outcome.

Secondary hyperparathyroidism – pathogenesis and the importance of serum parathyroid hormone values in CKD-MBD

The kidneys intervenes in calcium homeostasis by converting the storage form of vitamin D (25-hydroxyvitamin D3) through 1α hydroxylase enzyme, to the active form of vitamin D (1,25 dihydroxyvitamin D, calcitriol) that stimulates intestinal calcium absorption. It is was known that suppression of calcitriol synthesis occurs early in the evolution of CKD, before alterations in calcium, phosphorus and PTH can be detected[2] and thought to be caused by progressive reduction of renal mass function[2,8,9]. New studies changed this concept, by outlining that the suppression effect on 1 α hydroxylase exercised by circulating fibroblast growth factor 23 (FGF23) leads to a more important decrease in calcitriol synthesis than the loss of renal mass[10-12]. FGF23 is a hormone produced by osteocyte that expresses its

1 Fundeni Clinical Institute, Pediatric Nephrology Department

Therapeutics, Pharmacology and Clinical Toxicology6

action by a mandatory activating coreceptor named Klotho whose actions may be directly linked with vascular calcifications and therefore an increased mortality rate[21]. Their synergic action is to decrease renal proximal tubular phosphate reabsorption (that varies from 85% in normal kidney to under 15% in patients that have stage 4-5 CKD)[13] maintaining a normal phosphorus balance in early stages of CKD and to suppress the renal 1 α hydroxylase receptor, as a result of that lowering the synthesis of calcitriol (that will successively decrease gastrointestinal phosphate absorption)[14,15]. Several studies in children are consistent with data from the adult population and supports that [16-20] “increases FGF23 may be the first detectable biochemical evidence for an abnormal regulation of mineral ion homeostasis while glomerular filtration rate is merely normal, leading to the early decline in 1, 25(OH)2vitamin D levels”[2]. The decrease of calcitriol leads to an insufficiency of intestinal calcium absorption and as a result, the serum levels of PTH start to elevate[22]. This adjustment mechanism of PTH manages to maintain a normal calcium balance in early stage of CKD, by stimulating bone release. As a consequence of bone resorbtion though, it appears also an increasing level of serum phosphorus[2]. As long as the adaptive mechanisms and glomerular filtration rate are not severe damaged, a normal homeostasis can be maintained as a result of high levels of PTH. When glomerular filtration rate decreases, on the other hand, excretion of phosphorus is altered and hyperphosphatemia occurs causing a further suppression of calcitriol[23] and a stimulation of additional PTH release[24,25]. As kidney function reduces, appears an increasingly need of PTH levels in order to maintain an adequate mineral and bone metabolism. As a result of chronic exposure to high levels of PTH, accumulation of biologically active PTH fragments[27,28] and downregulation of the PTH receptor[28] we confront a resistance of the skeleton and kidney to the hormone action[26] and also a parathyroid hyperplasia which is difficult to reverse[24]. Hypocalcemia, hyperphosphatemia, lower levels of 1,25(OH)2D3 and changes in normal feedback of PTH due to a hyperplasic gland, have as result secondary hyperparathyroidism with a great impact on growth, development and long outcome of the children (bone deformities, fractures and soft tissue calcifications)[4, 30-34].

The importance of maintaining the serum levels of PTH in the optimal target ranges[2] depending on the stage of CKD is implied, considering its central role in the pathophisiology of this complex disease. Therefore, a detailed monitoring PTH along with calcium, phosphorus and alkaline phosphatase activity is recommended beginning with stage 2 of CKD in children[4]. Target levels of PTH depending on CKD staging are suggested. Unfortunately there is still an important controversy regarding the optimal levels of PTH in pre-dialysis CKD, and the “current recommendations by the National Kidney Foundation suggest that levels be maintained between 200 and 300 pg/ml[35], the European Pediatric Dialysis Group suggests that values between two- and threefold the upper limit are optimal in dialyzed

children[36], and a much broader range—between two- and nine times the range for normal individuals—is recommended by the Kidney Disease: Improving Global Outcome (KDIGO) foundation for patients treated with maintenance dialysis[4]”.

Abnormalities in bone turnover, mineralization, volume and linear growth in children

Bone disease in pediatric population with CKD has been a primary concern for physicians, because of the great impact that it has on a growing organism and in the sense of finding the most suitable treatments and better understanding of its physiopathology.

The most suitable tool that evaluates the bone disorder by three key histologic descriptors: bone turnover, mineralization, and volume, is the bone biopsy. Nevertheless, it is not routinely performed (because it does not predict the risk of fracture or type of osteodistropy), unless patients with CKD present pathological fractures,” persistent bone pain, unexplained hypercalcemia, unexplained hypophosphatemia, possible aluminum toxicity, and prior to therapy with bisphosphonates”[4].

The most frequent bone lesion that is observed in children with early stages of CDK is high bone turn-over, which is due to a high exposure of circulating PTH that activates excessively the osteoblasts and osteoclasts with consequences in bone structure and strength [37-39]. This is a cause for alterations in bone histology and may evolve to peritrabecular fibrosis or “osteitis fibrosa cystica”[40]. Over-suppression of PTH levels by excessive treatment or prolonged dialysis, on the other hand, has also a great impact on bone histology, in the sense of lowering the bone turn-over, a state named “adynamic renal osteodistrophy”. This condition is characterized by a lack of bone cellularity, a reduced bone formation rate, normal osteoid volume, a high risk for vascular calcifications (due to a high calcium and alkaline phosphatase levels) and severe growth impairment[2,41,42].

Mineralization of bone is another histologic parameter that is altered in chronic kidney disease. The mineralization of bone is essential for its hardness and strength, and its normal development depends on an elaborate process in which crystals of calcium phosphate are produced and placed in a well established architecture. If this process is not properly regulated, the result can be a concomitant increase in osteoid volume and osteoid maturation time3. The researchers have found that a central pawn in the correct mineralization is inorganic pyrophosphate, which inhibits abnormal calcification. Levels of pyrophosphate are controlled by at least three other molecules: nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), which produces pyrophosphate outside the cells; ankylosis protein (ANK), which transports it from the cell's interior to the cell surface; and tissue nonspecific alkaline phosphatase (TNAP), which decrease pyrophosphate in the extracellular environment, keeping its levels in control[43]. It is important to know that all this molecules are influenced by more than one biochemical parameter, which includes PTH, calcium, alkaline phosphatase, FGF-23[45,49] and also can be altered by medication that


we use with the intention to avoid disturbances of bone metabolism like active vitamin D sterols[44]. This is the reason why mineralization defects are high prevalent in pediatric CKD and is present even though the turn-over of the bone is normal, on early stages of CKD[45,46].

Children with chronic kidney disease have also abnormalities in bone volume, first of all because of the high levels of PTH which is an anabolic steroid with an impact on trabecular bone, leading to an increased bone volume, trabecular volume and width[47-48].

Another problem that has a severe impact on children’s outcome is linear growth failure. More than one third of CKD pediatric patients have a height deficit (under the 3rd percentile) even with a moderate renal function. This is caused by a multitude of factors like renal osteodistrophy, metabolic acidosis, chronic inflammation, difficulties in alimentation, functional hypogonadism and disturbances in endocrinal axe of insulin like GF1[4]. Guidelines strongly recommend the usage of human recombinant growth hormone (rhGH) therapy after resolution of malnutrition and biochemical abnormalities (PTH levels has to be monitored and controlled during therapy)[4], considering the degree of CKD, the growth potential and control of the osteodistrophy[2,50].

Cardiovascular consequences in CKD-MBD Even in early stages of CKD (when the first hormonal

systems to modify are FGF-23 and osteocalcin[11,53]) disturbances in mineral metabolism and loss of mesenchymal cell anabolism, influence the cardiovascular system, starting with a loss of vascular smooth muscle phenotype[51]. This leads to a decrease in vascular smooth muscle differentiation thus progressively losing its contractility[54,55]. This defect of contractility determines vascular stiffness, which is the cause of systolic high pressure and consequent left ventricular hypertrophy, an important manifestation in children with CKD[55]. Another effect of CKD-MBD is vascular calcification, in which a pathophysiological role may have vitamin D, FGF23 and hyperphosphatemia.

Different methods are used to assess the extent of cardiovascular damage in adults, but the methods still need to be standardized for the use in pediatric population. Carotid intima-media thickness and aortic pulse wave velocity are the most used methods to assess cardiovascular disorders in children, but they fail to describe vascular minimal changes in early stages of CKD[56].

Therapeutic agents in CKD-MBD The therapy in pediatric CKD-MBD has an important

role in controlling mineral metabolism, with the goal of improving linear growth, reducing bone fragility and deformities, increasing quality of life and reducing vascular calcifications[2].

The management includes maintaining serum calcium and phosphorus in normal limits, thereby maintaining appropriate PTH levels for CKD stage, preventing hyperplasia of the parathyroid glands, limiting extra-skeletal calcification and preventing the accumulation of toxics like ß2-microglobulin and aluminum[57].

Phosphorus control implies a combination of therapeutic agents like phosphate-binders and rigorous diet, in order to maintain its levels within the normal range, regarding the association of phosphorus and cardiovascular morbidity[57].

Phosphate binders can be classified in calcium-containing salts (calcium carbonate, calcium citrate and calcium acetate)- that are used to lower hyperphosphatemia and also as an additional source of calcium- and calcium-free phosphate binders (sevelamer hydrochloride, lanthanum carbonate, magnesium carbonate, stabilized polynuclear iron hydroxide and ferric polymaltose complex) that were developed to minimize the calcification risk and lowering the toxicity of calcium-containing salts[57].

Calcium-containing salts, as calcium carbonate, are proved to balance the phosphorus serum levels, by adjusting the dose proportional to the phosphorus content of the meal[34]. High doses, on the other hand, may lead to hypercalcemia (frequent in patients treated with vitamin D or adynamic bone disease[58]), an increase progression of vascular calcifications and rigidity and neurologic toxicity (aluminum hydroxide)

Sevelamer hydrochloride has the same effect on phosphorus levels as calcium-containing salts, but lowers the progression of vascular calcifications and improves the cardiac outcome by its action on cholesterol (lowers serum cholesterol levels and LDL cholesterol and increases HDL cholesterol) and has no side-effect as hypercalcemia. For preventing acidosis that may appear in treatment with sevelamer hydrochloride, has been introduced a new form named sevelamer carbonate[57,59]. Lanthanum carbonate is also a calcium-free phosphate binder, with the same effects as sevelamer, but because lanthanum is a heavy metal, has side effects as renal toxicity and accumulation in tissues and is not approved for children use[60].

Vitamin D therapy Low levels of 1, 25-dihydroxy vitamin D (calcitriol)

and hyperphosphatemia play a central role in secondary parathyroidism[30,61,62]. Providing adequate intake of vitamin D while controlling dietary phosphorus restriction has been shown to suppress successfully PTH levels[63,64].

KDOQI guidelines recommend vitamin D deficiencies to be treated with different doses of ergocalciferol or cholecalciferol depending of the stage of the deficiency, for 6 months[64].

Calcitriol and alfacalcidol express their effect on mineral metabolism by increasing the intestinal and renal calcium absorption, by increasing skeletal sensitivity for PTH and increasing FGF-23 levels, suppressing this way PTH levels, improve dialysed patients outcome, improve systolic pressure and reduce proteinuria and fibrosis in pre-dialysis patients[57,65,66]. This indirect mechanism of increasing FGF-23 levels may have consequences in time, by increasing cardiovascular risk[45]. Also, in association with calcium-based binders, active vitamin D sterols can lead in hypercalcemia and hyperphosphatemia, with an increased risk of tissue calcifications[57,67].


In order to minimize the side effect of hypercalcemia but maintaining a good impact on PTH levels, new molecules were developed: 22-oxacalcitrol, paricalcitol and doxercalciferol. Despite the beneficial effects on secondary parathyroidism, these new molecules seem not to improve bone mineralization[57]

CalcimimeticsCinacalcet is a molecule that activates calcium sensing

receptor, and it reduces PTH, FGF-23, phosphorus and calcium levels, inhibits parathyroid hyperplasia and controls bone mineralization[57,68]. Unfortunately there is still a lack of clinical trials in pediatric population with CKD in using calcimimetics, and the effects of this molecules on growth and cardiovascular protection are still to be investigated[58].

Parathyroidectomy for secondary hyperparathyroidism

Surgical parathyroidectomy is “the definitive therapy to manage uncontrolled”[70] secondary hyperparathyroidism and it is considered only when all medical therapies have failed. There are not well defined indications for this surgical intervention, especially in children, since cinacalcet therapy is largely used[71]. Nevertheless, KDOQI guidelines recommend parathyroidectomy in severe cases of hyperparathyroidism (iPTH > 88ɲmol/L) associated with hypercalcemia or hyperphosphatemia that are not responding to treatment, bone pathological fractures, vascular calcification or pruritus[63,70,71].

DiscussionChronic kidney disease and mineral bone disorder is

an important health problem, that affects an increasing number of pediatric patients, and has a powerful impact on child development and quality of life. New research bring to the front- line new molecules and hormones that are involved in mineral metabolism and cardiovascular health, new therapies are still undergoing clinical trials which makes us hopeful in treating this complex disorder. Also, an important thing to follow is starting more clinical trials in pediatric population, because there are striking differences between an adult organism and a growing one.

ACKNOWLEDGEMENT: This paper is supported by the Sectorial Operational Programme in Human Resources Development (SOP HRD), financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/159/1.5/s/137390/.

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Doina Anca Plesca21 Basarabia Blvd, Bucharest, Romaniae-mail: [email protected]

Abstract. Dystrophinopathies are X- linked muscle diseases, more frequently expressed in males, but infrequently also the substrate of muscular pathology in females. Laboratory methods in the diagnosis of dystrophinopathies have been continuously improving, which permits a higher accuracy in the study of the involved genetic alterations and of their modes of transmission and expression.Key words: dystrophin, Duchenne and Becker muscular dystrophies, X-linked pathology in women, X chromosome inactivation

Pop Anita1, Cornițescu M.1, Pleșca Doina Anca1

DIAGNOSIS PARTICULARITIES IN DYSTROPHINOPATHIES IN WOMEN

REVIEWTherapeutics, Pharmacology and Clinical ToxicologyVol XIX, Number 1, March 2015Pages: 12 - 18© Copyright reserved 2015

General data

Dystrophinopathies are caused by a functional deficit of dystrophin, a protein with an

essential role in the cell membrane binding of the actin-myosin complex. Clinically, dystrophinopathies cause impairment of the motor function, leading to respiratory deficit as well as skeletal deformities; they may be accompanied by cardiac and intellectual impairment. Initially described as muscle conditions affecting young boys, dystrophinopathies were linked to the dysfunction of the dystrophin protein, encoded by a gene located in the Xp21.2 region of the human X chromosome.

In the muscle fiber, dystrophin is located close to the internal side of the cell membrane as a component of a glycoprotein complex (the dystrophin-associated protein complex) [1] with a role in the anchoring of the actin-myosin myofibrils to the cell membrane. Dystrophin deficit leads to increased sarcolemmal deformability [2], one of the proposed explanation for the loss of muscle fibers in dystrophinopathies being that sarcolemmal lesions produced during muscle contractions favor the chronic necrosis of muscle fibers that becomes clinically manifest as muscle weakness.

Utrophin, a homologue of dystrophin, is overexpressed in dystrophinopathies [3]. It is not able to overcome the effects of the absence of dystrophin in human subjects, but its expression attenuates the effects of dystrophin deficiency in mdx mice [4].

The dystrophin gene is the largest identified in the human genome to date, comprising approximately 2.5 megabase in the Xp21.2 region of the human X chromosome. The gene consists of 79 exons; the presence of multiple alternative promoters that are differentialy expressed in tissues allows the tissue-specific messenger RNA synthesis. Several mechanisms enable the

generation of alternative transcripts from the dystrophin gene: the existence of three full-length transcription promoters (brain B, muscle M, Purkinje cell P), of internal promoters (retinal R, brain-3 B3, Schwann cell S, general G) and of an alternative splicing mechanism at the 3' end of the gene [5]. The transcript generated in the striated and cardiac muscle, under the control of the M promoter, includes all exons and is translated in the muscular dystrophin of 427 kDa molecular weight [6].

Clinical aspects in dystrophinopathies Clinical aspects vary in dystrophinopathies:

from mild, asymptomatic forms, with increased creatine kinase serum levels or with muscle cramps and myoglobinuria to severe forms that impair the function of skeletal and cardiac muscles (Duchenne and Becker muscular dystrophy) or the cardiac muscle alone (X-linked dilated cardiomyopathy) [7-10]. In what concerns the muscle impairment, three dystrophinopathy phenotypes are most prominent: Duchenne and Becker muscular dystrophies and the intermediate phenotype [11]

Duchenne muscular dystrophy (DMD) Initially described by the French physician

Guillaume- Benjamine Armand Duchenne at the end of the 1800s [12], DMD is the most frequent dystrophinopathy phenotype. DMD affects 1 in 3500 male newborns [13] and is caused by the total absence of the dystrophin gene expression [5]. The onset in boys occurs between two and three years of age and manifests itself as a delay in acquiring motor skills, or as a global developmental delay [11].

Muscle weakness is more pronounced proximally and manifests itself as a difficulty in climbing and running. The child falls more frequently than other children of same age, develops lumbar hyperlordosis and a waddling gait. A characteristic sign of the disease is the Gowers sign: climbing from the floor is possible by walking up one's own body. The pseudohypertrophy

1 “Carol Davila” University of Medicine and Pharmacy - Bucharest


of calf muscles can be identified [14].As the disease progresses, male patients lose

their motor skills and develop permanent muscle contractures, generalized muscle atrophy, defective thorax and spine conformation (kyphoscoliosis). They become wheelchair dependent generally by the age of 12 [10].

The fatigue of the diaphragm muscle and the pathological conformation of the thorax favour the frequent occurrence of respiratory infections and impede the cough effort. The respiratory condition requires adequate treatment, including mechanical ventilation [15].

The cardiac impairment manifests itself as primary dilated cardiomyopathy (DCM), conduction abnormalities and a variety of arrythmias, primarily supraventricular. The cardiomyopathy is characterized by extensive fibrosis of the posterobasal and eventually of the lateral free wall of the left ventricle [11].

Children suffering from DMD have a global developmental delay that is manifest from the first years of life. Qualitatively, they have a more severe impairment of verbal than non-verbal performances. Deficits in working memory and executive function have been found [10].

Male DMD patients do not transmit the dystrophin mutation [10]. Their health state seriously deteriorates before reaching the reproductive age.

The main cause of death of DMD patients is cardiorespiratory failure. Only few of them survive beyond the third decade [10].

Becker muscular dystrophy Becker muscular dystrophy (BMD) represents a less

frequent phenotype, with a milder impairment of the striated muscle but with frequent cardiac involvement [16]. It is generated by the presence in the skeletal muscle of either qualitatively abnormal or decreased quantity dystrophin [5].

The onset of Becker muscular dystrophy occurs at an older age than that of DMD, with milder clinical signs. Most patients preserve their motor skills by at least the age of 15, some remaining mobile at even older age [11].

Clinical signs include muscle weakness in the limb girdles, often calf pseudohypertrophy and activity-induced cramping in some individuals [10]. Muscle contractures and mental retardation are not as frequent or severe as in DMD [11].

In contrast to DMD patients, BMD patients have unaffected reproductive function, thus transmitting the BMD genetic trait to their daughters who will have a carrier phenotype. By not inheriting the X chromosome from fathers, their sons do not carry the dystrophin mutation [10].

Death generally occurs in the fifth decade due to heart failure [10].

The intermediate phenotype (outliers) The intermediate phenotype was described in

patients with disease signs that resembled either mild DMD of severe BMD forms. These patients become wheelchair dependent between the ages 12 to 16 [11].

Dystrophinopathies in women Unlike males with pathological alterations in

the dystrophin gene who are always symptomatic, females can be asymptomatic carriers or symptomatic individuals.

Asymptomatic carrier females have this status due to the inheritance of the dystrophin mutation on a single X chromosome and to the random X inactivation that occurs within the general female population. Although generally asymptomatic, they may reveal the presence of minor myopathic signs such as muscle cramps and myalgia. High creatine kinase serum levels are generally present [17].

Symptomatic women represent approximately one third of women with a pathological mutation in the dystrophin gene. 5% of them show myalgia and cramps without muscle weakness; 17% show mild to moderate muscle weakness while 8% show dilated cardiomyopathy, with a mean onset age of 33 [18]. A small fraction even show a severe muscular dystrophy phenotype that causes invalidity [19,20]. The onset of disease generally occurs after the age of seven [21].

The disease shows a slower progression in females than in males due to their expression of dystrophin in skeletal muscles at closer to normal levels as they age (genetic and biochemical normalization) [22].

The motor deficit in DMD women is of medium intensity, proximally located and affects especially lower limbs. Calf pseudohypertrophy is present [23].

Changes in muscle tone are less pronounced than in males with DMD; girls are able to walk until advanced ages, but they fall frequently and are unable to run. In rare cases they are wheelchair-bound [17].

Cardiac abnormalities are often found in women with dystrophin mutations. The most frequently reported are left ventricular dilation for both DMD and BMD female patients and dilated cardiomyopathy for DMD women. Studies are not concordant regarding the frequency of ECG abnormalities in women with dystrophin mutations [24].

Symptomatic girls also experience behavioural and mild learning abnormalities [17].

Pathophysiology of dystrophinopathies in women

In the event of a pathological mutation in the dystrophin gene, the dystrophinopathy phenotype can occur in women through several mechanisms:

1. By a translocation between the X chromosome and an autosome that disrupts the dystrophin gene on the X chromosome [25].

2. By the simultaneous presence of only an X chromosome (Turner syndrome; 45, X0 karyotype) and a dystrophin gene mutation on the only X chromosome in the genome [26-28].

3. By uniparental disomy of the X chromosome carrying the dystrophin gene mutation [29].

4. By the occurrence of a pseudohermafroditism in a person with female phenotype and male genotype (46, XY) that has on the only X chromosome in the genome simultaneous mutations in both the androgen receptor (AR)


gene and the dystrophin gene [30].5. By having simultaneous mutations in the

dystrophin gene alleles on both X chromosomes [19,31].

6. By preferential inactivation of the X chromosome without the dystrophin gene mutation (non-random X inactivation, skewed X-chromosome inactivation).

The mechanism of skewed X inactivation seems to be the most frequent cause of dystrophinopathies in women [20,32,33], the other mechanisms being detected only rarely.

X chromosome inactivation in women is definitive and usually random, taking place in the first mitoses in the human embryo [34].

Random X inactivation is a mechanism of balanced expression of genes on both X chromosomes, in the event of genetic mutations affecting the function of synthesized proteins. A dystrophin expression yield of 30% is sufficient to prevent the occurrence of muscle dystrophy [35].

Skewed X-chromosome inactivation is rare and generally due to some particularities of the regulating XIST center of inactivation on the X chromosome. The X chromosome inactivation is said to be skewed if more than 80% of a woman’s blood lymphocytes have the same parental X chromosome inactivated [36]. Although the X chromosome inactivation is assessed on a regular basis in the peripheral blood lymphocytes, the result of this test does not always correlate with the inactivation pattern in the muscle nuclei; a more accurate assessment of the X chromosome inactivation in muscles uses samples obtained from muscle biopsy [37].

Mutations in the dystrophin gene responsible for the occurrence of dystrophinopathies are large intragenic rearrangements (exon deletions and duplications) in 65-75% of cases; a smaller fraction of cases involve point mutations or small gene rearrangements [38,39].

In most patients, the clinical evolution can be predicted by using the reading frame rule. Thus, the majority of DMD patients have truncating mutations that lead to the absence of dystrophin expression, while BMD patients have mutations that preserve the reading frame, permitting the expression of semi-functional dystrophins [9,40].

Diagnosis of dystrophinopathies1. Diagnosis of dystrophinopathies in male patients

The diagnosis of muscle dystrophy is suspected based on clinical criteria: proximal distribution of muscle weakness; progressive decrease of force and generally a decrease of muscle volume; loss of osteotendinous reflexes; calf pseudohypertrophy; occurrence of the Gowers sign. A positive family history suggestive of X-linked inheritance strongly supports the suspicion of a dystrophinopathy.

Confirmation of the clinical diagnosis is based on biochemical, electrophysiological, histological, immunohistochemical and genetic assays.

The assessment of the muscular enzymes in serum reveals increased levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, aldolase, creatine kinase. The increase of the creatine

kinase levels, particularly of the MM isoenzyme, is the most sensitive and specific biochemical marker suggesting muscle lesion. Creatine kinase levels increase even in the early stages of the disease, prior to clinical signs; in advanced stages of the disease the serum levels of the enzyme decrease along with the decrease of the muscle volume. Higher values of enzyme serum concentrations are also found in asymptomatic carriers of the dystrophin mutations [17].

Electromyography shows myopathic changes.Muscle biopsy allows the identifying of the

histological aspects characteristic to muscle dystrophies as well as the quantitative assessment of muscle dystrophin synthesis, through immunohistochemical tests on the biopsy product or through Western Blot or ELISA assays on muscle tissue lysate.

The histological aspect specific to Duchenne and Becker dystrophies is represented by necrotic and regenerating muscle fibers that appear frequently in clusters, with abnormal fiber size variations, increase in the number of fibers with internal nuclei and replacement of the muscle tissue by connective and fat tissue [41].

Immunohistochemical tests on muscle biopsy samples reveal the absence of dystrophin expression and the overexpression of utrophin in Duchenne muscular dystrophy, while in Becker muscular dystrophy one can detect the presence of low level or qualitatively altered dystrophin [5].

Genetic diagnosis of dystrophinopathies

Several methods are available for the detection of mutations in the dystrophin gene that lead to the DMD and BMD phenotypes. The array of available diagnostic tools expanded towards higher resolution, allowing the detection of single nucleotide polymorphisms in the gene sequence.

Testing for the presence of dystrophin gene deletions and duplications Intragenic deletions and duplications represent the

main types of mutations identified in the dystrophin gene in children with dystrophinopathies [38,39]. Both qualitative and quantitative tests are available for testing these mutations.

Qualitative methods Multiplex PCR is a qualitative method for testing

the existence of deletions in the dystrophin gene that relies on simultaneous amplification in a PCR reaction of a set of gene exons, known to be most frequently affected.

Multiplex PCR primer sets for the dystrophin gene, described starting with the works of Beggs and Chamberlain [42,43] and then continuously improved, allow the detection of the most common deletions in the dystrophin gene. The method presents several disadvantages: it can confirm the existence of tested mutations but does not specify the ends of these since it does not test all gene exons; it cannot be used for the detection of deletions in the dystrophin gene in carrier women as it is not quantitative (the presence of the normal sequence in a dystrophin gene masks the existence of the mutation in the other allele).


Quantitative methodsQuantitative methods can be used in the diagnosis

of dystrophin mutations carrier women.Many quantitative methods are able to detect all

exon deletions and duplications, also specifying the ends of most rearrangements. Methods used in the past such as quantitative multiplex PCR of selected exons [44] or Southern blot hybridization of cell DNA with synthetic DNA probes [45] have been currently replaced by the optimized and commercially available method of multiplex ligation-dependent probe amplification (MLPA) [46].

A newer quantitative method for the high resolution testing of the dystrophin gene, including at the level of introns and splicing sites is the array comparative genomic hybridization (array CGH) [47-49]. This method employs sets of thousands of oligonucleotide sequences on solid support for hybridization with the corresponding sequences in the patient’s DNA genome, thus delivering data on the number of copies of the gene sequence along the entire 2.5 megabase dystrophin region, including all exons and introns. It maps the gene rearrangement points with a precision that depends on the selection of test probes in the respective set. A major advantage of the method is that, depending on the position in the gene sequence of the chosen probes, deletions and duplications are detected by several probes in the set (redundance of the obtained results).

Testing for the existence of small size mutations in the dystrophin geneA small proportion of mutations in the dystrophin

gene that induce dystrophinopathies are small size, including point mutations [38,39]. These mutations can be detected through gene sequencing [50], a currently highly accessible method.

Gene sequencing allows the identification of the entire nucleotide sequence of the assessed genetic material. The DNA material used in sequencing can be either genomic DNA extracted from the patient's tissue or complementary DNA obtained through reverse transcription from the patient's tissular messenger RNA. The obtained nucleotide sequences present differences depending on the nature of the assessed DNA material: genomic DNA contains both gene exons and introns while complementary DNA obtained by reverse transcribing the messenger RNA contains only exons.

Mutations identified in the complementary DNA must be confirmed in the genomic DNA. In some cases this step is difficult to perform: the cause of occurrence of mutations in the exon splicing regions in the messenger RNA may be located in the intronic sequence between the respective exons in the dystrophin gene. In such cases, the description of the modified sequence in the complementary DNA represents the information identifying the mutation [51].2. Diagnosis of dystrophinopathies in women

The diagnosis of dystrophinopathies in women benefits from the above-mentioned methods, also applicable in male patients.

The clinical suspicion of the presence of a dystrophin mutation in asymptomatic carrier women is generally raised by a family history of dystrophinopathy. In the

case of symptomatic women the trigger for diagnostic thinking is represented by the very signs of myopathy of the patient.

If within the family there are blood relatives with dystrophinopathy, their DNA material can be used as a comparative probe in the genetic testing. If test results from assays such as gene sequencing are already available for other affected members of the family, they can become referential for the women in study. Still, the identification of an anticipated mutation does not rule out other mutations in the person’s dystrophin genes.

In the absence of preliminary information on the dystrophin mutation of a female patient, previously mentioned tests can be applied successively, bearing in mind that the selected method should have the ability to detect the mutation in heterozygous state (in the presence of a corresponding normal allele in the genome).

Particularly in the case of women with dystrophinopathy it is useful to study the intrafamilial pattern of mutation transmission. Two methods are useful in this regard: the X chromosome segregation and the X chromosome inactivation analysis.

The X chromosome segregation analysis employs testing, with the aid of specific markers, of polymorphic regions on the X chromosomes in blood related members of the family and the evaluation of the inheritance pattern of the X chromosomes [52].

The X chromosome inactivation analysis may be performed by evaluating the X chromosome polymorphic sequences methylation. One version of this method is based on the discovery that the methylation of a HpaII restriction site located in the proximity of a variable length repetitive (CAG)n region in the X chromosome androgen receptor (AR) gene correlates with the X chromosome inactivation [53]. For each studied member of a family, the HpaII enzyme digestion of genomic DNA followed by PCR amplification may be used to determine the methylation status of parental X chromosomes. The quantitative result expressing the ratio between the amplified androgen receptor alleles provides the information regarding the X chromosomes inactivation pattern. In dystrophinopathies the study can be performed on DNA extracted from peripheral blood lymphocytes, but it is more relevant if performed on DNA extracted from muscle biopsy samples [37].

If non-random X chromosome inactivation is identified, the possible cause of the phenomenon can be evaluated by studying the non-coding XIST gene, whose RNA product is involved in the mechanism that determines the X chromosome inactivation [54]. Mutations in the promoter area of the gene have been associated with the non-random inactivation of the X chromosome [55].

A possible link has been described between skewed X chromosome inactivation and discordant X-related phenotypes in monozygotic twin girls. In such twin pairs, one girl presents the severe phenotype, while the other is only mildly affected or is clinically healthy. The phenomenon has been described for several X-linked diseases: Duchenne muscular dystrophy [56-60], clotting factor VIII deficiency (hemophilia A) [61,62] or clotting factor IX deficiency (hemophilia B, Christmas disease) [63]. The


mechanism of production of the discordant X-related phenotype in monozygotic twin girls is not yet fully understood.Conclusions

Dystrophinopathies are muscle diseases first described in males, caused by mutations in the dystrophin gene on the X chromosome. The clinical aspect of the disease in more variable and mild in women in comparison with that in males, mainly due to the high probability of inheriting another normal dystrophin allele in the genome and to the mechanism of random X chromosome inactivation in cell nuclei. Although the diagnosis benefits from the same methods as in men, supplementary tests for female patients are also available; these tests are useful for the study of the transmission patterns of the pathologic phenotype in families.

Disclaimer: The authors have no conflicts of interests

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Irina Magdalena DumitruClinical Hospital of Infectious Diseases Constanta, Romaniae-mail: [email protected]

Abstract. The evaluation of albendazol treatment’s efficacy in hydatid cysts (CH) with different locations and the evaluation of treatment’s efficacy based on: cysts’ location, size, type and immunological status of patient. We performed a prospective study on 320 patients, during 5 years. We performed the drug-based treatment in cases of small and medium cysts sizes (< 7 cm). Statistics do not show a significant difference between the hepatic and pulmonary hydatic cysts’ healing rates, p > 0.05 (p = 0.5987). Al-bendazol treatment’s efficacy is higher in the case of hepatic localization than in other localizations. The efficacy of the albendazol therapy is significantly higher in the case of CH < 7cm in comparison with CH > 7cm, which requests a surgical treatment. The efficacy of the albendazol treatment is higher in the case of type 1 hydatic cysts than in the case of type 2 hydatic cysts. Patients with CD4 > 944 cells/mm3 responded better to treatment in comparison with the patients with CD4 < 944 cells/mm3. The efficiency of the albendazol treatment is lower for the patients presenting co-morbidities. Treating hidatid diseases with albendazol 800mg/day for 4 weeks, in repeated cures separated by free intervals of 2 weeks, had as a result the healing of 91.25% of the cases. The positive predictive factors of the albendazol treatment response were: hepatic or pulmonary localization of the hydatid cysts, small dimensions, below 7cm in diameter, presence of the type1 hydatid cists, absence of co-morbidities and unaltered immunological status (CD4 > 944 cells/mm3).Key words: hydatid cysts, albendazol, efficacy

1. “Ovidius” University Constanta, Romania2. Clinical Hospital of Infectious Diseases Constanta, Romania

Dumitru Irina Magdalena 1,2, Dumitru E.1, Rugină S.1,2, Dumitru A.1

THE ALBENDAZOL TREATMENT’S EFFICACY IN HYDATID CYSTS

ORIGINAL PAPERTherapeutics, Pharmacology and Clinical ToxicologyVol XIX, Number 1, March 2015Pages: 19 - 22© Copyright reserved 2015

Introduction

Hydatid disease is a worldwide health problem [1-3]. Unfortunately, realistic national or

international figures do not exist for the total numbers of cases of cystic echinococcosis. The problem is that, the only basis for diagnosis was surgery, and few countries systematically reported cases. When they did report cases, uneven reporting occurred in different regions of countries. However, the increasing use of mass screenings with ultrasonography in endemic countries is generating important epidemiological data [4].

Two benzimidazolic drugs, mebendazole and albendazole, are the only anthelmintics effective against cystic echinococcosis. Albendazole and mebendazole are well tolerated but show different efficacy. Albendazole is significantly more effective than mebendazole in the treatment of liver cysts. Benzimidazole treatment alone requires prolonged administration over many weeks, with an unpredictable outcome in terms of response rates in individuals [5].

Objectives Primary: Evaluation of the albendazol treatment’s

efficacy in hydatid cysts with different locations,based to cysts’ location, size, type and immunological status of patient.

Secondary: Predictive response factors to drug-based treatment.

Material and methodWe performed a prospective study on 320 patients,

diagnosed with hydatid cyst in Clinical Hospital of Infectious Diseases Constanta, Romania, during 5 years (2008-2013). We performed the drug-based treatment in cases of small and medium cysts sizes (< 7 cm) or in inoperable patients (severe chronic diseases) with cysts size >7cm, with multiple cysts (2 or more organs), peritoneal cysts, patients with multiple reoccurrences.

Administration protocol for the Albendazol therapy: 800mg/day , for 4 weeks, in repeated cures separated by free intervals of 2 weeks [1-2].

Examinations’ calendar: 1. Initially, when included in the lot: - clinical examination; - APP (hydatid cyst, other chronic affections); - epidemiological context; - paraclinical evaluation: HLG, TGP, TGO,


serologic markers of the various types of chronic viral hepatitis (HBsAg, anti HCV, deltaAg, method - electrochemiluminescence (ECLIA), Roche Diagnostic, Switzerland;

- detection of anti Echinococcus granulosus antibodies (ELISA, NovaTec Immundiagnostica GmbH, Germany) through serologic reactions;

- Imaging evaluation - ECHO, x-rays, CT scans, MRI scans.

2. Monthly, during the treatment (before every therapeutic cure):

- clinical examination; - paraclinical evaluation: hepatic toxicity; - imaging evaluation – ECHO/x-rays (hydatic

cysts’ modifications due to treatment.3. At the end of the treatment: - clinical examination; - paraclinical evaluation: HLG, TGP,

TGO detection of anti Echinococcus granulosus antibodies (ELISA, NovaTec Immundiagnostica GmbH, Germany) through serologic reactions;

- imaging evaluation: ECHO/ x-rays, CT scan, MRI if needed.

4. ECHO/ X-rays and serological post-treatment surveillance, every 6 months for 2 years.

5. According to Gharbi classification (morphological classifications), we considered liver hydated cyst [6]:

a. Type I (CE1) - Pure fluid collection; b. Type II (CE2) - Fluid collection with a

detached membrane; c. Type III (CE3) - Fluid collection with multiple

septa and/or daughter cysts; d. Type IV (CE4) - Hyperechoic with high

internal echoes; e. Type V (CE5) - Cyst with reflecting calcified

thick wall.

ResultsDifferent locations were encountered, but the most

frequent occurrence was on the liver (67%), followed in frequency by the lungs (22%) . Other less frequent locations were the kidney, spleen, peritoneum, mediastinum, pericardium, brain, parotid gland, bone and muscle locations (1-3%).

Cysts dimensions between 3 and 7 cm were founded in most cases. The minimum cyst size was 1.1cm, while the maximum was 12 cm.

In most cases (90 patients), 6 therapeutic cures were necessary before the first healing signs appeared.

Patients’ healing rate was 91.25% while the causes of treatment failure were: complications (overgrowth that required surgery in 12 cases), albendazole toxicity (3 cases), non-response to albendazole treatment (6 cases), decease (one case with multiple lung cysts).

We compared the cases of healed patients with hepatic cysts with the cases of healed patients with pulmonary cysts. Statistics do not show a significant difference between the hepatic and pulmonary hydatid cysts’ healing rates (p = 0.5987) (Fig1).

The average of the cures followed for hydatid cysts with other localizations was 7.428571±1.886039.

Statistics show a significant difference between the hepatic or pulmonary and other locations hydatid cysts’ healing rates. We can conclude that the Albendazol treatment’s efficiency is higher in the case of hepatic or pulmonary localization than in other localizations. By this, we are saying that the hepatic and pulmonary localization represents a positive predictive factor for the therapeutic response (p = 0.009125) (Fig.2)

Most of the healed cases detained cysts < 7cm in size.

We can conclude that a dimension < 7 cm represents a positive predictive factor for the therapeutic response (Fig.3, Table I)

The average number of therapeutic cures administered

to the patients with type 1 cyst was 6.739 ± 1.91, and for the cases of patients with type 3 cysts it was 8.181 ± 2.39. We compared the two results, and the outcome was that

Figure 1.

Figure 2. Number of cures

112223455972230

0000000011921

0%10%20%30%40%50%60%70%80%90%

100%

Hepati

c

Pulmona

ry

Perito

neum

Spleen

Kidney

Muscle

Retroperi

toneu

mPer

icard Brai

nBone

Paroti

d

Medias

tinum

Healed patients Not healed patients

P=0.5987

Figure 3.


the number of therapeutic cures administered for the type 1 hydatid cyst (CE1) is significantly smaller than the number of cures administered for the type 3(CE3) hydatid cyst, thus the presence of the type 1 cysts being considered a positive predictive factor for the therapeutic efficiency (p<0.01, significant statistically).

The presence of comorbidities represented a negative predictive factor for the therapeutic response (Fig.4).

There were 2.8 % recurrences after a median delay of 16 months (12 – 24 months).

We performed CD4 count after therapy, for 20 responsive patients versus 19 non-responsive patients. Median CD4 for the two groups was 944 cells/mm3. Patients with CD4 > 944 cells/mm3 responded better to the treatment in comparison with the patients with CD4 < 944 cells/mm3 (p < 0.01). We conclude that CD4 represent a positive predictive factor for the therapeutic response.

DiscussionConstanta is an endemic area for Hydatidosis and

the Infectious Diseases Clinic has a long experience in therapy with albendazole. In the absence of systematic national reporting there is no recently epidemiological study and there are no reports about the effectiveness of therapy with albendazole in Romania. An international study conducted in 2009 from six centres (five countries including Romania, 711 treated patients ) reported that 1-2 years after the initiation of benzimidazole treatment, 75% of hydated cysts were classified as inactive, but the study was restricted to liver and peritoneal cysts [7]. Other authors have reported rates similar or lower than our study (91.25%): Gil-Grande 1993 – 94% [8], Teggi 1993– 77.9% [9], Franchi 1999 - 82.1% [10] and Keshmiri 2001 – 82% [11].

A recent study (Cappello E et al) on 30 patients

with hepatic hydatid cyst notified a cure rate of 76.6% and relapses were more frequent in patients treated with albendazole before 2005. Co-morbidities were observed in 17 (53.1%) patients (hypertension, chronic pulmonary diseases, hepatitis B and hepatitis C) and as in our study , they were a negative predictive factor for the therapeutic response and complications were observed in six patients (18.8%) these being cyst rupture, and bacterial infection [12].

In another study, albendazole was the most frequently used agent, the median duration of antihelminthic therapy was 6 months, as in our study, (range 0.7-144 months) and there were 17% recurrences after a median delay of 2 years (range 0.4-17 years). The presence of multiple visceral organ involvement increased the odds of recurrence by 5.4 (95% CI 3.1-9.4) [13]. In our study we reported only 2.8 % recurrences, but after a median delay of 16 months (10 – 24 months), in our protocol, we have recorded post-treatment surveillance every 6 months for 2 years. Only a Peruan study reported a low effectiveness of albendazole HCE therapy compared with the efficacy reported in clinical trials, 34% per patient and 40% per cyst after 3.8 ± 2.5 years [14].

In another chinese study, of 196 cases enrolled with liver hydatid cysts, after a long period with albendazole treatment (6 to 30 months) 32.7% were considered to be cured, 49% were improved, 14.3% remained unchanged, and 4.1% cases became aggravated. Patients with CE3 cysts needed a longer treatment course for cure (26.4 months), compared to cases with CE1 (20.4 months) or CE2 type (9 months) [15]. In our study the average number of therapeutic cures administered to the patients with CE1 cysts was 6.739 ± 1.91, and for the cases of patients with CE3 cysts it was 8.181 ± 2.39, more less than chinese study.

In relation to the role of CD4 in therapeutic response, in the last years, several studies have shown that genetic and immunologic host factors are responsible for the treatment resistance. Also in the adventitial layer surrounding the regressive and involutional hydatid cysts, infiltrating lymphocytes were composed mostly of CD4+ cells [16]. In our study CD4 was evaluated after treatment and probably a correct prediction analysis related to CD4 would have been possible if this value could have been measured prior to the initiation of therapy. We consider that CD4 counts can be a predictive factor for a response to treatment in patients, influencing the choice of optimal therapeutic intervention method.

ConclusionsTreating hydatid diseases with albendazol 800mg/day

for 4 weeks, in repeated cures separated by free intervals of 2 weeks, had as a result the healing of 91.25% of the cases (Fig.5-7).

The treatment has a similar efficacy in the hepatic and pulmonary hydatic disease (91.63%, respectively 88.88%).

The efficacy of the albendazol treatment is higher in the case of hepatic and pulmonary hydatic disease than in other localizations.

The positive predictive factors of the Albendazol treatment response were: hepatic or pulmonary

Table I.

Figure 4.

DIMENSIONS NUMBER OF HEALED CASES

< 7 cm < 3 cm 178 39 196 403 – 4.9 cm 85 875 – 6.9 cm 54 69

> 7 cm 7– 9.9 cm 21 17 30 22> 10 cm 4 8


localization of the hydatid cysts, small dimensions, below 7cm in diameter, presence of the type1 hydatid cysts, absence of co-morbidities and unaltered immunological status (CD4 > 944 cells/mm3).

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in hydatid cysts with different location” Thesis 20062. Dumitru I.M., Rugina S., Dumitru E. The

Albendazol Treatment’s Efficacy In Hydatic Cysts With Different Locations, EMOP, ORAL presentation, 2012

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5. Stojkovic M, Zwahlen M, Teggi A, Vutova K, Cretu CM, Virdone R, et al. Treatment response of cystic echinococcosis to benzimidazoles: a systematic review. PLoS Negl Trop Dis. Sep 29 2009;3(9):e524.

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8. Gil-Grande LA, Sanchez-Ruano J, Garcia-Hoz F, et al. Randomized controlled trial of efficacy of albendazole in intra-abdominal hydatid disease. Lancet 1993; 342: 1269-1272.

9. Teggi A, Lastilla MG, De Rosa F. Therapy of human hydatid disease with mebendazole and albendazole. Antimicrob Agents Chemother 1993; 37: 1679-1684

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11. Keshmiri M, Baharvahdat H, Fattahi SH, et al. Albendazole versus placebo in treatment of echinococcosis. Trans R Soc Trop Med Hyg 2001; 95(2): 190-194.

12. Cappello E, Cacopardo B, Caltabiano E, Li Volsi S, Chiara R, Sapienza M, Nigro L. Epidemiology and clinical features of cystic hydatidosis in Western Sicily: A ten-year review.World J Gastroenterol. 2013 Dec 28;19(48):9351-8.

13. Steinmetz S1, Racloz G, Stern R, Dominguez D, Al-Mayahi M, Schibler M, Lew D, Hoffmeyer P, Uçkay I. Treatment challenges associated with bone echinococcosis. J Antimicrob Chemother. 2014;69(3):821-6.

14. Salinas JL, Vildozola Gonzales H, Astuvilca J, Arce-Villavicencio Y, Carbajal-Gonzalez D, Talledo L, Willig JH. Long-term albendazole effectiveness for hepatic cystic echinococcosis. Am J Trop Med Hyg. 2011;85(6):1075-9.

15. Li T, Ito A, Pengcuo R, Sako Y, Chen X, Qiu D, Xiao N, Craig PS. Post-treatment follow-up study of abdominal cystic echinococcosis in tibetan communities of northwest Sichuan Province, China. PLoS Negl Trop Dis. 2011;5(10):e1364.

16. Riganò R, Buttari B, De Falco E, Profumo E, Ortona E, Margutti P, Scottà C, Teggi A, Siracusano A. Echinococcus granulosus-specific T-cell lines derived from patients at various clinical stages of cystic echinococcosis. Parasite Immunol. 2004 Jan;26(1):45-52.

Figure 5. Hydatid cysts before albendazol treatment

Figure 6. Hydatid cysts after 3 cures of albendazol treatment

Figure 7. Hydatid cysts after 6 cures of albendazol treatment


Elena Constanța Coleș1 I.C.Brătianu Blvd., 030171 Bucharest, Romaniae-mail: [email protected]

Abstract. Myeloproliferative neoplasms (MPN) are indolent hematologic cancers that are characterized by excessive production of mature blood cells. Acute myeloid leukemia (AML) is an uncommon, but often deadly complication of these chronic disorders. In the last years significant progress has been made in the diagnostic algorithm and therapeutic management of AML postMPN.Keywords: molecular genetics, mutations, myeloproliferative neoplasms, leukemic transformation, acute myelod leukemia

1. Colțea Clinical Hospital

Coleș Elena Constanța1, Stanca Oana1, Berbec Nicoleta1, Lupu Anca Roxana1

UPDATE ON MOLECULAR GENETICS IN ACUTE MYELOID LEUKEMIA POST MYELOPROLIFERATIVE NEOPLASMS

Therapeutics, Pharmacology and Clinical ToxicologyVol XIX, Number 1, March 2015Pages: 23 - 24© Copyright reserved 2015

Introduction

Myeloproliferative neoplasms (MPN) are a spectrum of clonal disorders of hematopoiesis

characterized by proliferation of mature-appearing myeloid cells and accumulation of bone marrow fibrosis [1]. MPN initiation and progression can not be explained by a single pattern of clonal changes, but rather by the acquisition of a wide panel of genetic anomalies.

The molecular biology of the BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) has witnessed unprecedented progress since the discovery of the acquired JAK2 V617F mutation in 2005, which was a turning point in the molecular diagnostic of these hematopoietic disorders [2].

But despite the high prevalence of JAK2V617F in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), and the common finding of dysregulated JAK-STAT signaling in these disorders, it is now appreciated that MPN pathogenesis can reflect the acquisition of multiple genetic mutations that alter several biologic pathways, including epigenetic control of gene expression (2). Genetic studies have identified recurrent somatic alterations in the majority of MPN patients, including acquired mutations in JAK2 (JAK2V617F) in 90% of polycythemia vera (PV) patients and ~50% to 60% of patients with essential thrombocytosis (ET) and primary myelofibrosis [3-6].

It has been long recognized that patients with myeloproliferative neoplasms (MPNs) are at significant, cumulative risk of leukemic transformation to acute myeloid leukemia (AML), which is associated with adverse clinical outcome and resistance to standard

AML therapies [7]. Leukemic transformation occurs in 8% to 23% of MF patients in the first 10 years after diagnosis and in 4% to 8% of PV and ET patients within 18 years of diagnosis and is associated with a universally poor prognosis [8-10].

Post-MPN AML is characterized by acquired somatic gene mutations, but, interestingly, mutations such as JAK2V617F, who appear to have a major etiologic role in the MPN, are sometimes absent in the AML clone, which means that a substantial proportion of patients with AML derived from a JAK2-mutant MPN are negative for JAK2 [11,12,13].

Studies showed that somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML, but not in chronic phase MPN and lead to clonal dominance of JAK2V617F/TP53-mutant leukemic cells [7].

Certain gene anomalies are identified at higher frequencies with disease evolution to the blast phase. The most frequent mutations involved in AML derived from a preexisting MPN were identified in TET2 , ASXL1 , IDH1 , and JAK2 [14-17].

In the majority of patients with JAK2-mutated MPN who progress to AML, the mutant clone is lost and TET2 mutations may be present in a clone distinct from that harboring a JAK2 wild-type cell. We could certainly affirm that in the pathogenesis of transformation to AML at least two different malignant clones are involved [18].

In contrast, ASXL1 mutations seemed to be detected in all phases of disease, and, most important, may precede the acquisition of JAK2 or even TET2 mutations in myeloblastic transformation [14,16].

Alterations in TET2 and ASXL1 can in some cases occur at an early stage of myeloid neoplasia, before the patient acquired a JAK2 mutation or even developed clinical evidence of MPN , which means that the acquisition of additional mutations leads to the development of clinically manifest disease phenotypes

THERAPEUTICAL PRACTICE


[14,15,16]. But although TET2/ASXL1 mutations may precede acquisition of JAK2 mutations by the MPN clone, mutations in TET2 and IDH1, but not ASXL1, are commonly acquired at the time of leukemic transformation [14,16,17].

ConclusionAlthough TET2, ASXL1, and IDH1 mutations have

been identified in patients with a spectrum of myeloid malignancies, the timing of acquisition of TET2/ASXL1/IDH1 mutations and the role of TET2/ASXL1/IDH1 mutations in progression from MPN to AML are not well known so far [14].

It seems that TET2 mutations are acquired after JAK2V617F or other myeloproliferative disorder disease alleles at the time of leukemic transformation of a JAK2V617F-positive or JAK2V617F-negative MPN clone. The mutational order of events in MPN and secondary AML varies in different patients, and TET2 and ASXL1 mutations have distinct roles in MPN pathogenesis and leukemic transformation, which maybe requires further research.

The presence of secondary AML that have no preexisting JAK2/TET2/ASXL1/IDH1 mutations, indicates the existence of other oncogenic alleles yet to be identified that are necessary for leukemic transformation [14].

All this progress made by genomic analysis of leukemic transformation occurred in MPN may have therapeutic implications who could improve the prognosis and outcome of these complex hematologic malignancies.

Conventional AML-style treatment appears to have limited efficacy in secondary AML emerging from MPN, although when coupled to allogeneic stem cell transplantation, some patients have long-term survival and prolonged DFS and OS (11). More likely, combinations of drugs that target different pathways will be required to develop successful therapeutic approaches in this disease. Less-intensive therapies/regimens such as hypomethylating agents appear to have significant clinical benefits in this group of patients. Combination of these agents with JAK1/ JAK 2 inhibitors, such as ruxolitinib, may add significant clinical benefits in some patients [19]. Also, both of these drugs seem to be well tolerated, with an acceptable toxicity profile, an important issue in the older age group [19].

More studies are needed in the future in order to emphasize the impact of genetic features on outcome and thus to optimize the therapeutic responses.

References1. Campbell PJ, Green AR. The myeloproliferative

disorders. N Engl J Med 2006;355:2452–66. Medline Google Scholar

2. Nguyen HM, Gotlib J. Insights into the molecular genetics of myeloproliferative neoplasms. Am Soc Clin Oncol Educ Book. 2012:411-8.doi: 10.14694/ EdBook_AM.2012 .32.411.

3. Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia

with myelofibrosis. Cancer Cell. 2005;7(4):387–97.4. Baxter EJ, Scott LM, Campbell PJ, et al.

Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365(9464):1054–61.

5. James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434(7037):1144-8.

6. Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005;352(17):1779–90.

7. Rampal R, Ahn J, Abdel-Wahab O, Nahas M, et al. Genomic and functional analysis of leukemic transformation of myeloproliferative neoplasms. Proc Natl Acad Sci USA. 2014 ; Dec 16;111(50).

8. Gaidano G, Guerrasio A, Serra A, Rege-Cambrin G, Saglio G. Molecular mechanisms of tumor progression in chronic myeloproliferative disorders.Leukemia. 1994;8 (Suppl 1) :S27–9.

9. Mesa RA, Li CY, Ketterling RP, Schroeder GS, Knudson RA, Tefferi A. Leukemic transformation in myelofibrosis with myeloid metaplasia: a single-institution experience with 91 cases. Blood. 2005;105(3):973–7.

10. Najean Y, Rain JD. The very long-term evolution of polycythemia vera: an analysis of 318 patients initially treated by phlebotomy or 32P between 1969 and 1981. Semin Hematol. 1997;34(1):6–16.[PubMed]

11. Heaney ML1, Soriano G. Acute myeloid leukemia following a myeloproliferative neoplasm: clinical characteristics, genetic features and effects of therapy. Curr Hematol Malig Rep. 2013 Jun;8(2):116-22.

12. Campbell PJ, Baxter EJ, Beer PA, et al. Mutation of JAK2 in the myeloproliferative disorders: timing, clonality studies, cytogenetic associations, and role in leukemic transformation. Blood. 2006;108(10):3548–55.

13. Theocharides A, Boissinot M, Girodon F, et al. Leukemic blasts in transformed JAK2-V617F-positive myeloproliferative disorders are frequently negative for the JAK2-V617F mutation. Blood. 2007;110(1):375–9.

14. Abdel-Wahab O, Manshouri T, Patel G, Harris K, et al. Genetic Analysis of Transforming Events That Convert Chronic Myeloproliferative Neoplasms to Leukemias. The Journal of Cancer Research. 2010.70;445.

15. Delhommeau F, Dupont S, Della Valle V, et al. Mutation in TET2 in myeloid cancers. N Engl J Med. 2009;360(22):2289–301.

16. Carbuccia N, Murati A, Trouplin V, et al. Mutations of ASXL1 gene in myeloproliferative neoplasms. Leukemia. 2009.

17. Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med. 2009;361(11):1058–66.

18. Beer PA, Delhommeau F, LeCouédic JP, et al. Two routes to leukemic transformation after a JAK2 mutation-positive myeloproliferative neoplasm. Blood. 2010 Apr 8; 115 (14) :2891-900.

19. Eghtedar A, Verstovsek S, Estrov Z, Burger J, et al. Phase 2 study of the JAK kinase inhibitor ruxolitinib in patients with refractory leukemias,including postmyeloproliferative neoplasm acute myeloid leukemia. Blood. May 17, 2012; 119 (20).


Madalina Marilena Oprea Coltea Clinical Hospital 1-3 I.C. Bratianu Blvd, 030171 Bucharest, Romaniae-mail: [email protected]

Abstract. Autoimmune thrombocytopenia is a common immune hematologic complication associated with lymphoproliferative diseases, most frequent with chronic lymphocytic leukemia and non-Hodgkin’s Lymphomas . The relation between autoimmune cytopenias and lymphoproliferative malignancies is well known and the mechanisms are complex. Regarding the ethiology, one or more simultaneously oc-curring causes could leed to thrombocytopenia. We should make a very riguros clinical and paraclinical judgement in diagnosis of autoimmune thrombocytopenia and then, treat not only the number of platelets but also manage the risk of significant bleeding. Supportive care , corticosteroids and chemo-imunotherapy are the currently used treatment , individualized for each pacient. The response to the lymphoma treatment is very good, and significant bleeding is rare.Keywords: imune thrombocytopenia, lymphoproliferative diseases, antiplatelets antibodies

1. Hematology Department - Colțea Clinical Hospital, Bucharest2. “Carol Davila” University of Medicine and Pharmacy, Bucharest

Oprea Mădălina Marilena1, Ivănescu Ana-Maria1, Coliță A.1,2, Turbatu A.1, Coleș Elena2, Lupu Anca Roxana1,2

THE MANAGEMENT OF THE AUTOIMMUNE THROMBOCYTOPENIA ASSOCIATED WITH CHRONIC MALIGNANT LYMPHOPROLIFERATIVE DISEASES


Introduction

Chronic malignant lymphoproliferation are associated with autoimmune thrombocytopenia

(ITP) 1-5% in chronic lymphocytic leukemia (CLL) [1, 2], 0,76% in Non-Hodgkin Lymphoma (NHL) [3], 0,29% in Hodgkin Lymphoma (HL) [4].

CLL is the most often lymphoproliferative disease associated with autoimmune disorders . ITP complicate the course of limphoproliferative diseases, but also may precede the diagnosis ( in CLL flow citometry of older pacients with ITP releved CD19/C5+ lymphocytes)[1,2].

Heterogenous histopatologycal forms of NHL are more frequent associated with ITP like: diffuse large B-cell lymphoma (DCLL) , small lymphocytic lymphoma, marginal zone lymphoma, waldenstrom disease/ myeloma, follicular lymphoma, mantle cell lymphoma; T cell lymphomas and lymphomas after autologous stem cell transplantation [3].

In Hodgkin's disease ITP take place most frequent in active phases or in periods of complete remission. The most important characteristics of the pacients are: old age , advance disease and non - nodular sclerosing histology[1, 4].

Ethio - pathogenesis Immune thrombocytopenia is a complex disorder

of immune disregulation wich cause the autoantibody production[5, 6, 7]. The pacient underlying condition could perturbed the immune system before the onset of ITP , but also a pacient with one autoimmune disease is a high risk to develop a second [8] (was found CD 19/CD 5 + clones in bone marrow from older CLL patients with ITP)[9].

Thrombocytopenia may reflect a part of immunological imbalance, closely related to the pathophysiological background of lymphoproliferative disease (HL, NHL)[4, 5]. In some cases the platelet antibody could be produced by lymphoma tissue itself (was demonstrated IgM platelet antibodies in lymphoplasmacytic lymphoma[3].

Beside the implication of B and T cells in auto-antibodies production (CD8+ cytotoxic T-cells from patients with active ITP antibodies bound and lysed platelets in vitro [10], another components of the immune system are disturbed : the reticuloendothelial system, receptors on monocytic phagocytic cells [3,5,7,8] (was found increased levels of BAFF = B cell activating factor, part of the tumor necrosis factor family [7,11]), and the complement (was observed complement-dependent lysis of platelets in vitro)[10].

It was observed that the autoantibody production in secondary ITP has the same mechanism of platelet destruction as in primary ITP [1] and include: production of antiplatelet antibodies by B lymphocytes against 2



The occurrence of stereotyped configuration of B cell receptor (BCR) - HCDR3 (subsets 1 and 7) was associated with shorter time to ITP development compared to other patients[17]. Early appearance of ITP in the course of CLL and refractoriness to treatment were associated with the poorest outcomes[15]. The overall survival of patients with autoimmune cytopenia as a whole was not significantly different from the patients without this complication[18].

In CLL patients – in contradiction to autoimmune hemolytic anemia , ITP is not view like a complication of advanced disease[1,15]. Severe autoimmune thrombocytopenia is not correlate with active CLL disease[1]. ITP in CLL patients is associated with poor survival, but independent of the common clinical prognostic variables[16].

In Hodgkin lymphomas, pacients who presented with autoimmune cytopenias had a particular demographic and disease - related profile in contrast with the other, (not differ significantly from those who did not associated ITP)[4, 19].

ITP therapyThere is currently no consensus for a therapeutic

approach to lymphoma - ITP[20]. The treatment in secondary ITP is often the same to the one used for primary ITP, but the management must be focused to obtaine a complet remission of the lymphoid malignancy and not to treat the decreased platelet number[1]. Indication for treatment: platelets <20/30.000/mm3, active bleeding or high bleeding risk associated with platelets < 50.000/mm3[5,6].

There are several parameters that must take into consideration (beside the platelet number): age, bleeding symptoms, health-related quality of life, side effects associated with therapy, the urgency of chirurgical procedures: simple or complex extraction ( the platelet count recommended are > 30/50.000 /mm3) , minor or major surgery ( >50/ 80.000 / mm3)[5, 6].

Traditional first-line therapy of ITP include corticosteroids, intravenous imunoglobuline (IVIG ) and imunoglobuline antiRh ( D ) (the last two treatment are rare used in current practice - mainly due to high cost). Corticosteroids (Prednisone 1-2mg/ kg /day) for 4 weeks are the standard initial treatment in primary or secondary ITP[5, 14].

• In CLL response to first-line treatment, usually steroids or IVIg, is ~50 % to 60 %; about 20% are refractory [15]. The immunosupresion of this treat-ment could increased morbidity and mortality due to infections in CLL. Rituximab, monoclonal antiC-D20antibody is known to be active in both CLL and ITP, needs to be further investigated in ITP associated with CLL[2,5].Vincristine is effective in some cases given at 1 mg weekly for 4 to 6 weeks[15].

• In Hodgkin lymphomas - ABVD combination chemotherapy (Doxorubicin, Bleomycin, Vinblastine, Dacarbazine) provided effective control of HL and the autoimmune condition[3,4].

• In NHL patients, prednisolone alone is often inef-

groups of glycoproteins: GPIIbIIIa and / or the GPIbα, expressed on platelets and also on megakaryocytes, • peripheral autoimmune destruction of platelets by

increased platelets removal and phagocytosis by splenic macrophages,

• reduced bone marrow megakaryocyte production [6,8] by suppression of maturation , increased phago-cytosis , alteration in megakaryocyte apoptosis , and

• disturbances in metabolism of trombopoietin (TPO) and its receptor, c-Mpl. These are the regulators of megakaryocytes and platelets production, with im-portant roles in the differentiation of hematopoietic stem cells [12] (levels of TPO plasma are not elevated in patients with ITP) [10].DiagnosisIt is considered that the diagnosis of autoimmune

thrombocytopenia is more a diagnosis of exclusion[5,6]. If thrombocytopenia appear in the course of the lymphoproliferative disease it can have simultaneously multiple causes, like: chronic hypersplenism/ splenomegaly, bone marrow infiltration or/and autoimmunity, but also recent treatment – chemotherapy (especially the use of purine analog - Fludara) or heparin treatment[2, 9].

The international working group recommend platelet value of < 100.000/mm3 on peripheral blood count for the diagnosis of immune thrombocytopenia[2,3,5].

In CLL - thrombocytopenia is associated with IV stage (RAI classification) - secondary to bone marrow infiltration . It could be considered immune mediated when appear a rapid (< 2 weeks), important (half of initial number) and “unexplained” fall in platelet count [2,9], associated with the absence of splenomegaly on physical examination and no cytotoxic treatments in the last month[13].

There is no “gold standard” test that can reliably establish the diagnosis . [14] Platelet antibody detection test is not recommended at first intention because it shows an increased variability, and poor sensibility (platelets - associated IgG (PaIgG) are elevated in both immune and non-immune thrombocytopenia)[5,14]. Beside malignant lymphoproliferative disease the pacient coud have also immune conditions like chronic infections (HIV and hepatitis B, C) and autoimmune diseases (ex. systemic lupus erythematosus )[2,5]. In this regard it is necessary to thoroughly analyze the clinical and laboratory dates . Some tests have potential utility: glycoprotein specific antibody, antinuclear, antiphospholipid and antithyroid antibodies, a positive direct red-cell antiglobulin test [6,14]; another uncertain benefit: serum trombopoietina, PaIgG, bleeding time, platelet survival, serum complement[14].

Proper diagnosis and treatment of the underlying disorder play an important role in patient management.

The relation between ITP and common prognostic factors

The development of ITP in pacients with CLL was associated with a high white blood cell count (WBC), a positive DAT, unmutated genes of immunoglobulin heavy chain ( IgVH ) , ZAP70+ [2, 15, 16].


fective, and chemotherapy for the lymphoma is recommended . The corticosteroids and Rituximab are included in most imunochemotherapy regimens used in NLH or in CLL: COP (Ciclofosfamida, Vin-cristin, Prednison)/ and /or Doxorubicina (CHOP) / Etoposide (COEP) and /or Rituximab (R) with good recovered of ITP after lymphoma treatment [3,5].The second line therapy include splenectomy,

rituximab-monoclonal antiCD20antibody and thrombopoietin - receptor agonists (TPO-R): Romiplostim and Eltrombopag[5].

In primary ITP splenectomy was considered the only “curative”therapy, but it side effects like sepsis, infections or possible vascular complications determine that pacients and doctors don’t accept splenectomy as therapy for ITP[5]. In NHL - ITP was observed that splenectomy is effective in small lymphocytic and splenic marginal zone lymphomas, with a partial response in DLCL lymphoma[3].

The role of Rituximab (one dose/month or 4 doses/month) was studies in many randomized trials, recognized its application in many autoimmune conditions. In CLL has been used with good results [5,15]. Was reported high response rates using as initial therapy the combination of Rituximab with high-dose dexamethasone[14]. Is not clearly defined the most effective dose , a recent study in non - CLL - associated ITP has found that a lower dose of 100 mg weekly was effective[15]. Previous response to corticosteroids, secondary ITP and shorter duration of ITP, could be predictors of response to Rituximab[5].

Romiplostim and Eltrombopag are TPO - R agonists and increase the circulating platelet count by stimulation of platelet production by the bone marrow (BM) megakaryocytes[5,6,14]. Most studies include adults pacients with primary ITP, refractory to conventional treatment, only a few dates discuss the role of thrombopoietin receptor agonists in ITP associated with CLL [9,21]. Using Romiplostin was limited the use of steroids in severely immunocompromised patients; also was mentioned his usefulness for the determination of the histological diagnosis of the lymphoma through a safe and unmodified tissue sample biopsy [28].

Both TPO- R agonists have been associated with decreased bleeding events, reduced need for additional medications, and improved quality of life. The most important side effect are the rebound of thrombocytopenia upon abrupt drug discontinuation, the risk of BM reticulin formation and thromboembolic events [6,16]. Importantly, the cost/benefit balance should also be considered when using a TPO - R agonist[20].

Conclusions

Autoimmune thrombocytopenia is one of the cause of thrombocytopenia who can be associated with the malignant lymphoproliferative diseases. The perturbation of the immune response is a complex process , an important role have the auto-antibodies production against platelets and megakaryocytes. The

diagnosis is more one of exclusion, and we have a lot of tests who help us for diferentions. About the therapy, it must take into account more parameters, not only platelets values: age, the risk of severe bleeding, the side effects of the treatement and the necessity of majore and / or urgency surgery. The lymphoma chemo-imunotherapy have a good response about autoimmune thrombocytopenia. Adding novel treatment is a true challenge if quality of life is not impaired.

References1. Liebman HA. Recognizing and treating secondary

immune thrombocytopenic purpura associated with lymphoproliferative disorders. Semin Hematol. 2009 Jan;46(1 Suppl 2):S33-6.

2. Visco C., Ruggeri M., Evangelista M. L. Impact of immune thrombocytopenia on the clinical course of chronic lymphocytic leukemia. Blood. 2008 111: 1110-1116.

3. Hauswirth A.W., Skrabs C., Schützinger C. et al. Autoimmune thrombocytopenia in non-Hodgkin’s lymphomas. Haematol. March 1, 2008; 93 ( 3): 447-450.

4. Burghuber OC, Smolen J, Kühböck J. Immune thrombocytopenia in Hodgkin's disease. Acta Med Austriaca. 1986;13(2):55-9.

5. Neunert. C.E. Current management of immune thrombocytopenia. ASH Education Book. December 6, 2013; vol 2013 no. 1: 276-282.

6. Cuker A. and Cines D. B.Immune Thrombocytopenia. December 4, 2010; 2010 (1) : 377-384.

7. Cines, D. B., Bussel, J. B., Liebman, H. A., & Luning Prak, E. T. (2009). The ITP syndrome: pathogenic and clinical diversity. Blood, 113(26), 6511–6521. doi:10.1182/blood-2009-01-129155.

8. Johnsen Jill. Pathogenesis in immune thrombocytopenia: new insights. ASH Education Book December 8, 2012 ; vol. 2012 ( 1) : 306-312.

9. Hodgson K.,Ferrer G., .Moreno C. Chronic lymphocytic leukemia and autoimmunity: a systematic review. Haematol. May 1, 2011; vol. 96 ( 5):752-761.

10 . Kistanguri G and McCrae K. R. Immune Thrombocytopenia. Hematol Oncol Clin North Am. Jun 2013; 27(3): 495–520.

11. John W. Semple. Platelets & Thrombopoiesis. ITP has elevated BAFF expression. Blood. December 17, 2009; 114 (26).

12. Cines D. B, Liebman H., Stasi R. Pathobiology of secondary immune thrombocytopenia. Semin Hematol. Jan 2009; 46 (Suppl 2) :S2-14

13. Visco C. Rodeghiero F. Immune Thrombocytopenia in Lymphoproliferative Disorders. 2009 December; Volume 23(6): 1261–1274

14. Provan D. Stasi R. Newland A.C. International consensus report on the investigation and management of primary immune thrombocytopenia. Blood. January 14, 2010; 115 (2)

15. Dearden C. Disease - Specific Complications of Chronic Lymphocytic. Leukemia ASH Education Book January 1, 2008 vol. 2008 no. 1 450-1456.


16. Advani R.H, . Zelenetz A. D, Abramson J. S. Non-Hodgkin's Lymphomas J.Natl. Compr. Canc. Netw. 2011;9:484-560.

17. Maura F, MD, Visco C., Novella E. Biology and Pathophysiology, excluding Therapy: Poster II Immune Thrombocytopenia in Patients with Chronic Lymphocytic Leukemia Is Associated with Stereotyped B-Cell Receptors Blood ASH Annual Meeting Abstracts 2011.

18. Moreno C., Hodgson K., Ferrer G. et al. Autoimmune cytopenia in chronic lymphocytic leukemia: prevalence, clinical associations, and prognostic significance. Blood. December 2, 2010; 116 (23).

19. Kalpadakis Christina. Autoimmune hemolytic

anemia and autoimmune thrombocytopenia at diagnosis and during follow-up of Hodgkin lymphoma. Leukemia & lymphoma Impact Factor. 2.61. 01/2012; 53(8):1481-7. DOI: 10.3109/10428194.2012.660628 .

20. Al - Nawakil C. Park S. Chapuis N. Salvage therapy of Autoimmune Thrombocytopenic Purpura revealing non-Hodgkin Lymphoma by the thrombopoietin receptor agonist romiplostim. British Journal of Haematolog. January 2012; Vol. 156 (1): 145–147.

21. Eltrombopag for the Treatment of Immune ThrombocytoPenia (ITP) Secondary to Chronic Lymphoproliferative Disorders (LPDs). ClinicalTrials.gov. Last review September 2012.


Ioana Petrescu Coltea Clinical Hospital 1-3 I.C. Bratianu Blvd, 030171 Bucharest, Romaniae-mail: [email protected]

Abstract. Residual lithiasis found in the main bile duct entails the presence, at the site, of one or more calculi, consequent to a surgical procedure for gallbladder or common bile duct lithiasis. Currently, the surgical community is trying to treat this condition via a minimally invasive procedure: endoscopic retrograde cholangiopancreatography. A bile duct obstacle can be diagnosed based on several imagistic methods, the most important ones being: the ultrasound, computed tomography or the cholangio- MRI. These methods aim to detect an obstacle, to establish its nature, location and size. The decision to apply these imagistic diagnostics techniques or different ones, depending on their indications, contraindications or their succession belongs, entirely, to the radiologist. In this sense, we compared the results we have obtained so far to specialised studies and research, in an attempt to establish the highest reliability level in the diagnostic imaging investigation of the common bile duct lithiasis whose absence would make impossible an endoscopic retrograde colangiopancreatography.Keywords: choledocholithiasis; ultrasonography; computed tomography; magnetic resonance cholangiopancreatography; endoscopic retrograde colangiopancreatography

1. “Coltea” Clinical Hospital, “Carol Davila” University of Medicine and Pharmacy, Bucharest2. “Floreasca” Emergency Clinical Hospital, “Carol Davila” University of Medicine and Pharmacy, Bucharest

Petrescu Ioana1, Bratu Ana Magdalena1, Popa B.V.2, Cristian D.1, Petrescu S.1, Zaharia C.1, Burcos T.1

THE RADIOLOGIST’S ROLE IN THE MINIMALLY INVASIVE THERAPY OF RESIDUAL COMMON BILE DUCT LITHIASIS


Introduction

The restant choledocolithiasis represents the residual calculi in the main bile duct, following

a procedure at the level of the biliary tract, in history up to a maximum of two years. The approach of a patient that presents restant calculi in the common bile duct relies significantly on the radiologist, who will give a precise diagnosis and localization of the lithiasis as well as provide guidance to the surgeon in view of applying a minimally invasive therapy - ERCP.

Finding an obstacle in the common bile duct requires an immediate and precise location, the establishment of the structure, the assessment of the obstruction degree as well as possible proximal modifications, either pre-existent ones or caused by the calculus itself. Hence, having a clear-cut image of these details depends on imaging methods. In this sense, the most essential data are provided by the following exploration techniques: ultrasound, computed tomography (CT) or cholangio-MRI(MRCP).

Our study aimed to compare current literature with results from our clinical experience regarding the most

effective imaging means to diagnose a restant common bile duct lithiasis that grounds a minimally invasive therapy, as well as their selective use based on benefits and disadvantages.

Imaging methods In order to visualise the bile duct, an ultrasound is

the first step taken following the clinical examination. In a limited number of cases an abdominal ultrasound shows the bile duct calculi. In most situations, ultrasounds evince only the dilated bile duct. If the images are inconclusive, this technique will guide the radiologist to opt for a different exploration method, which, in fact, constitutes its utility.

A computed tomography (CT) stands for an imagistic exploration method that detects the presence of an obstacle in the bile duct; it provides an estimation of the obstructed segment and sets the cause of the blockage as well as ensures extra data on the simultaneous abdominal pathologies.

The quality of a CT examination increases along with an augmented number of slices that lead to high resolution sections and numerous post processing possibilities.

A cholangio-MRI (Mangentic resonance cholangiopancreatography MRCP) is the most sensitive method that permits an in-depth analysis of the bile duct without contrast agent, identifying with 90-95% sensitivity, the presence of an obstacle in



lithiasic lesions possibly responsible for the dilatation of the biliary tract, a CT examination with intravenously administered contrast agent is mandatory. A tumoral lesion will grow in density by contrast overflow, while a calculus will keep the same density [10].

In 87% of billiary lithiasis cases, Maurea has found a correspondence between the data obtained from multislice computed tomography (MSCT) and MRCP [2].

Anderson has found moderate sensitivity and specificity degrees of the native CT followed by intravenous contrast administration, by using axial sections when detecting choledocholithiasis [10]. Tseng revealed that CT diagnosis has been significantly lower in patients with common bile duct calculi smaller than 5 mm (56.5%) compared to patients that presented choledocholithiasis larger than 5 mm (81.2%), the coronal plane reconstructions exerting no modification in the CT’s efficacy of diagnosing the common bile duct lithiasis [11].

In terms of evincing choledocholithiasis Pickuth discovered 86% sensitivity and 98% specificity of the native CT, this procedure being useful especially for the cases of calculi at the level of the ampulla [6].

Our own experience revealed values of MRCP sensitivity and specificity above 95% in the procedure of choledocholithiasis diagnosis. All patients diagnosed with this condition were further exposed to ERCP, as a minimally invasive therapy thus obtaining a 100% rate of lithiasic jaundice cure. Kalra reconfirms that magnetic resonance cholangiopancreatography (MRCP) is the only non-invasive diagnostic method for billiary obstruction, with a high potential to replace endoscopic retrograde colangiopancreatography

the bile duct. The technique doesn’t rely on exposure to X-rays but, unlike the CT, it cannot highlight the nature of the obstruction. However one of the most essential traits is its effectiveness in tracing even the smallest calculi that usually do not appear during a CT examination.

Results We focused and studied the cases of all patients in

Coltea Clinical Hospital, Bucharest who underwent ERCP for restant bile duct lithiasis.

The patients with excision of the gallbladder that presented to our clinic for jaundice, subsequently to the clinical exam were submitted to an abdominal ultrasonography.

Our experience with ultrasound in patients with mechanical jaundice and prior ablation of the gallbladder emphasized the dilation of the billiary tract in 98% of cases. A precise individualization through ultrasound of the bile duct lithiasis was achieved for 72% of the overall analysed cases.

Compared to our results, literature expands and describes the experience of various specialists in the field.

Terhaar demonstrated an 86,4% negative predictive value of the ultrasound coming to the conclusion that if an ultrasound detects a main bile duct calculus with a dimension larger than 10mm without an obvious cause, the following step will most likely be an MRCP [1].

A smaller calculus, if positioned in the distal common bile duct will be missed by the ultrasound examination. In this context, Maurea proved that if a calculus has been localised in the distal common bile duct this happened through magnetic resonance cholangiopancreatography (MRCP) and not through an ultrasound [2].

According to Ferrari, if there is any clinical and laboratory suspicion of common bile duct lithiasis, with no confirmation from the abdominal ultrasound, the patient has to undergo an MRCP. On the other hand, if the clinical and lab data raised the main bile duct calculus suspicion and the ultrasound confirmed the lithiasis then the patient will either suffer an endoscopic retrograde cholangiopancreatography (ERCP) or will undergo surgery [3].

However, among the community many support the ultrasound as the first imagistic choice in the assessment of patients with suspicion of billiary obstruction, like Ghimire [4] or others who will opt for subsequent investigation techniques- Blackbourne [5] and Pickuth [6,7].

Kristiansen sustains that patients with cholangitis, jaundice and choledocholithiasis detected via ultrasound will benefit directly from ERCP [8].

In what concerns the computed tomography (CT), our experience proved a 95% accuracy of the method that detects a calculus and over 95% rate of success in terms of precise localization. Once detected, the billiary obstruction must be etiologically differentiated: benign-malignant.

Rosch found 77% sensitivity and 63% specificity of the CT during the process of diagnosing the billiary strictures [9].

In order to obtain a comprehensive image of non

Figure 1a. Ultrasound -lithiasis CBD

Figure 1b. CT- biliary ducts lithiasis


(ERCP) - as the first assessment technique of the bile duct obstruction [12]. Hou sustains that CT and MRI explorations of the biliary tract are less invasive diagnostic methods that need to be cautiously applied prior to the interventional endoscopy [13].

Literature contains numerous studies on MRCP’s sensitivity, specificity, positive and negative predictive values in the diagnosis of choledocholithiasis. However these traits vary from study to study as in Griffin’s and Norero’s: sensitivity- 84% and 97%; specificity- 96% and 74%, negative predictive value- 93% and 90%, positive predictive value- 91% and 91% [14, 15]. In Taylor and Topal’s research sensitivity scored 97,9% and 95%, specificity- 89% and 100%, positive predictive value- 83,6% and 100% while negative predictive value scored 98,6% and 98%. [16, 17].

In Pisani’s comparative study on ERCP-MRCP, one of the main conclusions was that the two methods have similar sensitivity when distinguishing a biliopancreatric obstruction [18]. In the same context, Sakai obtained data that support MRCP as replacement of ERCP, namely as a more effective method to detect choledocholithiasis [19].

In a different comparative study on magnetic resonance cholangipancreatography, endoscopic retrograde colangiopancreatography and ultrasound Maurea concluded that MRCP is superior in detecting the calculi at the level of an intrahepatic bile duct (IHBD) and of an extrahepatic bile duct (EHBD). [2]

Maccioni believes that MRCP can be mainly involved in the diagnosis of biliary obstruction. When necessary, it is doubled by a conventional MRI with enhancing agent in the area of the upper abdomen

and, possibly, by functional studies, thus generating a morphological and functional “all in one” study on the biliopancreatic tree [20].

Conclusions 1. Despite its numerous deficits in what concerns

the diagnosis of the main bile duct lithiais, the ultrasound remains, for most cases, the first investigation in patients with mechanical jaundice, especially due to its low costs and lack of counter-indications. The most recognised merit is the easy visualisation of the biliary tree dilatation, thus offering a wide perspective of the following diagnostic phases, in view of highlighting the calculi and resolving the case.

2. A CT examination distinguishes with a higher accuracy than an ultrasound the remaining bile duct calculi in patients submitted to cholecystectomy. The biggest advantage of a CT is the precise localization and provider of clear data on the calculus structure. If contrast medium is used intravenously it makes the difference between the nature of the biliary tract obstacle- benign or malignant, with a complete description of the possible associated proximity tumour processes.

3. MRCP is the proper investigation method for the biliary tree, with the highest diagnostic value in the case of a choledocian obstruction. Although it cannot distinguish between benign and malignant features it will provide accurate data to the surgeon who will apply ERCP for billiary stone extraction.

4. The succeeding diagnostic step, after an ultrasound, should depend on the radiation dose the patient is submitted to (children, pregnant women, women in their fertile age), the counter-indication related to the contrast agent and an MRI exam. All these aspects enter and constitute the radiologist’s responsibility.

5. ERCP represents the top therapeutic choice for the removal of biliary calculi. It is the least traumatizing method for patients with obstructive jaundice by choledocholithiasis. Also, the method allows the palliative percutaneous drainage in the tumour pathology.

6. ERCP requires a precise imagistic diagnostic that renders the radiologist indispensable for the minimally invasive therapy, needed in the choledocian lithiasis removal procedure.

Author's contribution: All authors contributed equally to the manuscript.

References1. Terhaar OA, Abbas S, Thornton FJ, Duke D,

O'Kelly P, Abdullah K, Varghese JC, Lee MJ.- Imaging patients with "post-cholecystectomy syndrome": an algorithmic approach. Clin Radiol. 2005 Jan;60(1):78-84.

2. Maurea S, Caleo O, Mollica C, Imbriaco M, Mainenti PP, Palumbo C, Mancini M, Camera L, Salvatore M. - Comparative diagnostic evaluation with MR cholangiopancreatography, ultrasonography and CT in patients with pancreatobiliary disease. Radiol

Figure 2a. MRCP choledocholithiasis

Figure 2b. ERCP bile duct stone extractions


Med. 2009 Apr;114(3):390-402. doi: 10.1007/s11547-009-0374-x.

3. Ferrari FS, Fantozzi F, Tasciotti L, Vigni F, Scotto F, Frasci P. -US, MRCP, CCT and ERCP: a comparative study in 131 patients with suspected biliary obstruction. Med Sci Monit. 2005 Mar;11(3):MT8-18.

4. Ghimire R, Lohani B, Pradhan S- Accuracy of ultrasonography in evaluation of level and cause of biliary obstruction: a prospective study. Kathmandu Univ Med J (KUMJ). 2005 Jan-Mar;3(1):17-21.

5. Blackbourne LH, Earnhardt RC, Sistrom CL, Abbitt P, Jones RS.- The sensitivity and role of ultrasound in the evaluation of biliary obstruction. Am Surg. 1994 Sep;60(9):683-90.

6. Pickuth D, Spielmann RP- Detection of choledocholithiasis: comparison of unenhanced spiral CT, US, and ERCP. Hepatogastroenterology. 2000 Nov-Dec;47(36):1514-7.

7. Pickuth D.- Radiologic diagnosis of common bile duct stones. Abdom Imaging. 2000 Nov-Dec;25(6):618-21.

8. Kristiansen VB, Rosenberg J, Kehlet H.- Diagnosis of common bile duct stones in symptomatic gallstone disease. Ugeskr Laeger. 2000 Jul 31;162(31):4134-9.

9. Rösch T, Meining A, Frühmorgen S, Zillinger C, Schusdziarra V, Hellerhoff K, Classen M, Helmberger H.- A prospective comparison of the diagnostic accuracy of ERCP, MRCP, CT, and EUS in biliary strictures. Gastrointest Endosc. 2002 Jun;55(7):870-6.

10. Anderson SW, Lucey BC, Varghese JC, Soto JA.- Accuracy of MDCT in the diagnosis of choledocholithiasis. AJR Am J Roentgenol. 2006 Jul;187(1):174-80.

11. Tseng CW, Chen CC, Chen TS, Chang FY, Lin HC, Lee SD.- Can computed tomography with coronal reconstruction improve the diagnosis of choledocholithiasis? J Gastroenterol Hepatol. 2008 Oct;23(10):1586-9. doi: 10.1111/j.1440-1746.2008.05547.x.

12. Kalra M, Sahani D, Ahmad A, Saini S. -The role of magnetic resonance cholangiopancreatography in patients with suspected biliary obstruction. Curr Gastroenterol Rep. 2002 Apr;4(2):160-6.

13. Hou LA, Van Dam J.- Pre-ERCP imaging of the bile duct and gallbladder. Gastrointest Endosc Clin N Am. 2013 Apr;23(2):185-97. doi: 10.1016/j.giec.2012.12.011.

14. Griffin N, Wastle ML, Dunn WK, Ryder SD, Beckingham IJ.- Magnetic resonance cholangiopancreatography versus endoscopic retrograde cholangiopancreatography in the diagnosis of choledocholithiasis. Eur J Gastroenterol Hepatol. 2003 Jul;15(7):809-13.

15. Norero E, Norero B, Huete A, Pimentel F, Cruz F, Ibáñez L, Martínez J, Jarufe N. -Accuracy of magnetic resonance cholangiopancreatography for the diagnosis of common bile duct stones. Rev Med Chil. 2008 May;136(5):600-5. doi: /S0034-98872008000500008.

16. Taylor AC, Little AF, Hennessy OF, Banting SW, Smith PJ, Desmond PV. -Prospective assessment of magnetic resonance cholangiopancreatography for noninvasive imaging of the biliary tree. Gastrointest Endosc. 2002 Jan;55(1):17-22.

17. Topal B, Van de Moortel M, Fieuws S, Vanbeckevoort D, Van Steenbergen W, Aerts R, Penninckx F. - The value of magnetic resonance cholangiopancreatography in predicting common bile duct stones in patients with gallstone disease. Br J Surg. 2003 Jan;90(1):42-7.

18. Pisani JC, Bacelar A, Malafaia O, Ribas-Filho JM, Czezcko NG, Nassif PA.- Comparative study between magnetic resonance cholangiopancreatography and endoscopic retrograde cholangiopancreatography in the diagnosis of the pancreatic and biliary obstruction. Arq Gastroenterol. 2001 Jul-Sep;38(3):149-57.

19. Sakai Y, Tsuyuguchi T, Tsuchiya S, Sugiyama H, Miyakawa K, Ebara M, Saisho H, Yokosuka O.- Diagnostic value of MRCP and indications for ERCP. Hepatogastroenterology. 2007 Dec;54(80):2212-5.

20. Maccioni F, Martinelli M, Al Ansari N, Kagarmanova A, De Marco V, Zippi M, Marini M.- Magnetic resonance cholangiography: past, present and future: a review. Eur Rev Med Pharmacol Sci. 2010 Aug;14(8):721-5.

21. Stroszczynski C, Hunerbein M.- Malignant biliary obstruction: value of imaging findings. Abdom Imaging. 2005 May-Jun;30(3):314-23.

22. Soto JA , Castrillón GA. Clinical applications of magnetic resonance cholangiopancreatography. Radiologia. 2007 Nov-Dec;49(6):389-96.

23. Chu YL, Wang XF, Gao XZ, Qiao XL, Liu F, Yu SY, Zhang J.- Endoscopic ultrasonography in tandem with endoscopic retrograde cholangiopancreatography in the management of suspected distal obstructive jaundice. Eur J Gastroenterol Hepatol. 2013 Apr;25(4):455-9. doi: 10.1097/MEG.0b013e32835ca1d7.

24. Ascunce G, Ribeiro A, Rocha-Lima C, Larsen M, Sleeman D, Merchan J, Szabo D, Levi JU- Single-session endoscopic ultrasonography and endoscopic retrograde cholangiopancreatography for evaluation of pancreaticobiliary disorders. Surg Endosc. 2010 Jun;24(6):1447-50. doi: 10.1007/s00464-009-0798-3.

25. Kaltenthaler EC, Walters SJ, Chilcott J, Blakeborough A, Vergel YB, Thomas S - MRCP compared to diagnostic ERCP for diagnosis when biliary obstruction is suspected: a systematic review. BMC Med Imaging. 2006 Aug 14;6:9.

26. Al-Mofleh IA, Al-Rashed RS, Al-Amri SM, Al-Ghamdi AS, Al-Faleh FZ, Al-Freihi HM, Isnani AC.- Malignant biliary strictures. Diagnosis and management. Saudi Med J. 2003 Dec;24(12):1360-3.

27. Di Cesare E, Puglielli E, Michelini O, Pistoi MA, Lombardi L, Rossi M, Barile A, Masciocchi C- Malignant obstructive jaundice: comparison of MRCP and ERCP in the evaluation of distal lesions. Radiol Med. 2003 May-Jun;105(5-6):445-53.

28. Sharma SK, Larson KA, Adler Z, Goldfarb MA.- Role of endoscopic retrograde cholangiopancreatography in the management of suspected choledocholithiasis. Surg Endosc. 2003 Jun;17(6):868-71.

29. Caddy GR, Tham TC.- Gallstone disease: Symptoms, diagnosis and endoscopic management of common bile duct stones. Best Pract Res Clin Gastroenterol. 2006;20(6):1085-101.


Ion Ștefan1 Calistrat Grozovici Str., sector 2, Bucharest, Romaniae-mail: [email protected]

Abstract. Malaria, whose history is almost as old as humanity’s itself, constitutes a permanent challenge and reason of concern for the medical community. The involvement of the fifth plasmodium species – Plasmodium knowlesi has become an undeniable reality. Also, expanded choloroquine resistance, mefloquine resistance, the occurrence of artemisinin resistance as well as the lack of an efficient vaccine, the possibility of P. vivax transmission to persons with negative Duffy antigen, resistance of vectors to DDT and derivates represent matters of high relevance for the scientists in the field. However, these threats led to intensified efforts to discover new therapeutic and diagnosis breakthroughs.Keywords: malaria, Plasmodium knowlesi, RDTs, Tafenoquine, Pironaridine, RTS,S vaccine

1. ”Dr. Carol Davila” Central Universitary Emergency Military Hospital, Bucharest – Infectious Diseases Ward

Ștefan I.1

MALARIA – A CONTINUOUS CHALLENGE. UPDATES AND NEW PERSPECTIVES ON THE DIAGNOSIS AND TREATMENT OF MALARIA


Having high mortality and morbidity rates, malaria stands as the most important human

disease which, based on World Health Organization’s and CDC’s 2013 estimates, affected 198 million people, generating approximately 584,000 deaths annually. That is 1 death every two minutes, mostly in African children (in Tropical Africa, 1 out of 20 children dies before the age of five). Currently there are five pathogen plasmodium species in humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi. The zoonotic potential of P. knowlesi, a simian originally parasite, was considered limited (very few cases of infection in humans) until 2004, when in Sarawak, Malaysia several human infection cases were recorded. Since then, more cases have been registered, especially in South-Eastern Asia which ultimately led to the recognition of P. knowlesi as the fifth pathogen plasmodium in humans. The lifecycle of P. knowlesi takes 24 hours, triggering, in a short amount of time, high parasitemia, hence the disease’s degree of severity. Microscopic diagnosis can be challenging as young trophozoites morphologically resemble those in P. falciparum, while the final erythrocytic stages of the parasite cannot be differentiated from P. malariae. Given these similarities, P. knowlesi infections are often microscopically misdiagnosed as P. malariae infections. Bearing in mind the severity of P.

knowlesi infection [1,2,3,4] which can be fatal, WHO established that any microscopically obtained result reported as P. malariae, should be considered positive for P. knowlesi or P. malariae in all the areas where the former was identified (Borneo. Combodgia, Myanmar, The Phillipnes, Singapore, Thailand) [5].

The sensitivity of optical microscopy in detecting infections at the level of the placenta is quite reduced [6], a high risk being that malaria infections during pregnancy are associated with major adverse effects as anaemia [6,7] and low birth weight [8]. In order to facilitate the diagnosis, the scientific community recommends the extended use of rapid diagnostic tests (RDTs), as screening tests for pregnant women who live in endemic areas for malaria [9,10]. Out of these endemic areas, rapid diagnostic tests are used by travellers for self diagnosis purposes, when they return from malaria endemic areas. This type of tests were used in Great Britain, proving 97% sensitivity and 95% specificity, compared to microscopy [11]. Recently, FDA has approved the use of a mixed rapid diagnostic test – BinaxNOWMalaria – that combines antibodies that detect both HRP 2 (histidine-rich protein 2) – antigen for P. falciparum, and aldolase – a common enzyme in all plasmodium species, whose serum level correlates with parasitemia. Used on a lot of 256 travellers that presented fever, the test showed 94% sensitivity and 84% specificity [12].

A novelty is the occurrence of P. vivax infection in negative Duffy antigen populations in Eastern and western Africa, in Madagascar or those around the Amazon [13-19].

In terms of treatment we must mention that WHO’s recommendations insist on combined



therapies with artemisinin, especially in the case of P. falciparum. However, recently five countries in Mekong subregion (Cambodia, Laos, Myanmar, Thailand, Vietnam) have reported cases of artemisinin resistance; other such cases were found at the border between China and Myanmar. The most probable causes of resistance occurrence seem to be artemisinin based monotherapy and incomplete treatment (premature discontinuation once symptomatology disappears). Presently, WHO advises testing resistance to antimalarial drugs in these areas.

For pregnant women that live in high-transmission areas, WHO’s endorsements in 2012 pleaded for sulfadoxine-pyrimethamine therapy, administered at every prenatal check-up, during the second and third trimester. According to the World Health Organization, prenatal visits must take place as follows: first visit during the first trimester (no chemoprophylaxis at this time), the second visit between weeks 24 and 26, a third visit at 32 weeks and a final visit during weeks 36-38 of pregnancy [20]. Similarly, for children in Africa, below the age of 5, season antimalarial chemoprophylaxis is recommended (monthly, during the rainy season) with amodiaquine + sulfadoxine-pyrimethamine, administrated together with the vaccines in the regular immunization schedule.

A new molecule is being evaluated both as P. falciparum infection prophylaxis as well as curable drug for P. vivax infection. The molecule under discussion is tafenoquine - an 8-aminoquinoline, a structural analogue of primaquine. It is active against hepatic stages of P. falciparum and P. vivax (for the latter being effective against hypnozoites too) and efficacious against asexual erythrocytic stages or gametocytes. Its half-life is longer than primaquine’s but just the same, it can induce haemolytic anaemia in patients with glucose-6-phosphate dehydrogenase deficit. Currently, a study is ongoing to assess Tafenoquine single dose’s efficacy, in combination with chloroquine. [21]

Another promising molecule is pyronaridine – hydroxyaniline-benzo-naphthyridine. It is active in vivo and in vitro against chloroquine-resistant species. Still, there are concerns regarding a possible resistance development when used in monotherapy [22-23]. Hence, when treating P. falciparum malaria it is recommended to use it in combination with artemisinin [24]. This combination displayed similar efficacy as artemether- lumefantrine – the most used artemisinin combination – or as artesunate and mefloquine combined therapy [25-26]. Although considered a drug with reduced adverse events, some cases with pyronaridine induced hepatotoxicity have been reported [27]. Despite this, its main advantages are the single daily dose and a different chemical structure than other antimalarial drugs, ultimately constituting a backup therapeutic option for cases of resistance to commonly used antimalarial drugs.

Staying in the therapeutic field, lately, the usefulness of adjusting primaquine doses depending on the plasmodium species invloved has been esthablished. In order to prevent schizogony reviviscence in P. vivax or P. ovale infections, chloroquine treatment must

be associated with primaquine administration for 14 days. If in the case of P. ovale 15 mg/day X 14 days dosage turned out efficacious, for P. vivax infections the same dose generated 10% relapses [28]. In a trial on British tourists, recently returned from South Asia and diagnosed with P. vivax malaria, 30mg/day X 14 days dose proved superior, in terms of efficacy. The doubling of doses also prevents the occurence of P. vivax primaquine resistant strains.

In Cambodia, Artemether-lumefantrine the optimal therapy in most parts of the world, failed. The association between artesunat and amodiaquine registered 10% higher failure rates in 6 out of 23 studies deployed in African States and Indonesia. For these cases, dihydroartemisinin-piperaquine combination, highly efficacious in Mekong subregion, could be a solution.

The costs of therapy, occurrence of multidrug resistance, significant side effects, counter indications and compliance issues urged the need for an effective antimalarial vaccine. The ideal vaccine should be affordable, safe, induce long term immunogenicity – if possible life immunogenicity – and active in all plasmodium forms. Despite constant preoccupation, numberless studies and research, these criteria are difficult to meet, due to Plasmodium species’ high rate of multiplication, increased antigenic variability, complicated structures and complex life cycle. During the 1990s the first attempts to produce a malaria vaccine belonged to Elkin Patarroyo who, by using circumsporozoite protein (CS) as antigen – the most important surface antigen of the pre-erythrocytic stage – obtained a low immunogenicity and high reactogenicity vaccine that turned out inefficient in Africa. In 2012, GlaxoSmithKline (GSK) produced, by genetic engineering, the RTS,S/AS01 vaccine, containing P. falciparum circumsporozoite protein, a part of the surface antigen of HBV virus and a chemical adjuvant for the amplification of the immune response. In phase III studies, the vaccine exhibited modest results therefore, in October 2013, GSK formulated the RTS,S vaccine, that was submitted to the European Medicines Agency in July 2014, in order to be approved and licensed, that being expected during the second half of 2015.

References1. Cox-Singh J, Davis TM, Lee KS, Shamsul SS,

Matusop A, Ratnam S, Rahman HA, Conway DJ, Singh B. Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening. Clin. Infect. Dis. 2008; 46:165–171.

2. Cox-Singh J, Hiu J, Lucas SB, Divis PC, Zulkarnaen M, Chandran P, Wong KT, Adem P, Zaki SR, Singh B, Krishna S. Severe malaria—a case of fatal Plasmodium knowlesi infection with post-mortem findings: a case report. Malar. J. 2010;9:10. doi:10.1186/1475-2875-9-10.

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4. Barber BE, William T, Grigg MJ, Menon J, Auburn S, Marfurt J, Anstey NM, Yeo TW. A


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9. Tagbor H, Bruce J, Agbo M, et al. Intermittent screening and treatment versus intermittent preventive treatment of malaria in pregnancy: a randomised controlled non-inferiority trial. PLoS One 2010; 5:e14425.

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12. Farcas GA, Zhong KJ, Lovegrove FE, et al. Evaluation of the Binax NOW ICT test versus polymerase chain reaction and microscopy for the detection of malaria in returned travelers. Am J Trop Med Hyg 2003; 69:589.

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negative individual. Malar J 2011; 10:336.18. Culleton R, Ndounga M, Zeyrek FY, et al.

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21. Llanos-Cuentas A, Lacerda MV, Rueangweerayut R, et al. Tafenoquine plus chloroquine for the treatment and relapse prevention of Plasmodium vivax malaria (DETECTIVE): a multicentre, double-blind, randomised, phase 2b dose-selection study. Lancet 2014; 383:1049.

22. Ringwald P, Bickii J, Basco LK. Efficacy of oral pyronaridine for the treatment of acute uncomplicated falciparum malaria in African children. Clin Infect Dis 1998; 26:946.

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25. Tshefu AK, Gaye O, Kayentao K, et al. Efficacy and safety of a fixed-dose oral combination of pyronaridine-artesunate compared with artemether-lumefantrine in children and adults with uncomplicated Plasmodium falciparum malaria: a randomised non-inferiority trial. Lancet 2010; 375:1457.

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2. Signature of all authors3. Manuscript - the text of the manuscript should be submitted as a single document4. References5. Tables and/or figures


Manuscript preparation

All articles must be original material, not previously published and not currently under consideration for publication in another journal. Each paper, including the abstract and references may not exceed 10 pages. The paper should be written in English. Text should be supplied in a format compatible with Microsoft Word (with .doc extension). All manuscripts must be typed left justified only with margins of at least 2,5cm, at double space, in 12 points, Times New Roman or Arial font for normal text and symbol font for all symbols. Indent first line at 10mm. All text should be black-or-white. Do not use colored text. Avoid using „Styles and Formatting” of MS Word.

TITLE: not exceeding 50 characters (including spaces), it should be brief but informative. No abbreviations allowed.

ABSTRACT: a brief summary of the research; include a brief introduction, a description of the tested hypothesis, the approach used to test the hypothesis, the recorded result and the conclusion. No abbreviations allowed, cited.

KEYWORDS: 5-6 specific word, not repeating the title of the manuscript.

TEXT: the text of the original research manuscript should be arranged in the following sections – introduction, materials and methods, results, discussion, conclusions. Introduction: description of the research area, relevant background information, and the hypotheses tested in the study should be included. Materials and methods: materials and subjects utilized in the study as well as the procedures undertaken to complete the work. The methods should be described in sufficient detail if they could be repeated by a competent researcher. Mentioning of the statistical tool used for analysis of the results. All procedures involving experimental animals or human subjects must be accompanied with a statement on the necessary ethical approval from an appropriate ethics committee. Results: should describe the reason for each experiment, the obtained result and its significance. Discussion: relate the results’ section to the current understanding of the scientific problem being investigated; discuss the significance of your result. Avoid the use of footnotes in the text. Use the metric system, SI units to express weight and measures. Clearly indicate the place where the table or figure is to be inserted.

TABLES AND FIGURES: insert title above the table with initial capitals, extra space but no punctuation. They should be numbered in the order of their mentioning in the text. Use Roman numerals for tables and Arabic numerals for figures. Table should be in MS Word. Figures: most figures are in black-and-white, without gridlines. Save as .tiff, .jpeg. The minimal accepted resolution – 300 dpi. Do not place a box or ruled frame around a finished table. Place figures and tables separately, at the end of manuscript, one per page. Any table or figure must have a legend. For graphs Excel graphs are acceptable. Present 2D data and avoid 3D plots.

ACKNOWLEDGEMENTS: can mention people who have helped make your manuscript possible. Mention all applicable grants and other funding that supported your work.

REFERENCES: number consecutively in the order that are cited in the text not in alphabetical order. The list must contain the first six authors (if there are least six) or the first three authors (if more than six) and et all. Articles: authors should be presented: last name, initial of first name. Indicate title of article, name of journal, year of publication, volume and issue number, start-end page. Journal name abbreviation should conform to those used in Index Medicus. If journal is not included in Index Medicus use the entire title of the journal. For articles published in online journals also add the URL and the date when the article was accessed. Books: author of the chapter, title of the chapter, editor, title of book, edition, city and country, publisher, year, specific pages. For web contents list: author, title of the page or content, name or owner of the web site, URL, publication, update, access date. It is the responsibility of the author to verify the accuracy of all references.

ABBREVIATION, ACRONYMS AND SYMBOLS: use only standard abbreviations. Abbreviations must be defined on their first use in the paper – the full term should be spelled out first, followed by the abbreviated term in parentheses.

The article should be submitted to Therapeutics, Pharmacology and Clinical Toxicology

Editorial Office: Dr. Cristina Negulescu, INBI „Prof. dr. Matei Bals”, Pavilion IV, Etaj IVStr. Dr. Calistrat Grozovici nr. 1, Sector 2, București, CP 021105 or by e-mail at the address: [email protected]


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