Genotype replacement of the human parainfluenza virus type 2 in Croatia between 2011 and 2017 - the role of neutralising antibodies.

Previously we reported on the HPIV2 genotype distribution in Croatia 2011-2014. Here we expand this period up to 2017 and confirm that G1a genotype has replaced G3 genotype from the period 2011-2014. Our hypothesis was that the G1a-to-G3 genotype replacement is an antibody-driven event. A cross-neutralisation with anti-HPIV2 sera specific for either G1a or G3 genotype revealed the presence of genotype-specific antigenic determinants. By the profound, in silico analyses three potential B cell epitopic regions were identified in the hemagglutinin neuraminidase (regions 314-361 and 474-490) and fusion protein (region 440-484). The region identified in the fusion protein does not show any unique site between the G1a and G3 isolates, five differentially glycosylated sites in the G1a and G3 genotype isolates were identified in epitopic regions of hemagglutinin neuraminidase. All positively selected codons were found to be located either in the region 314-316 or in the region 474-490 what indicates a strong positive selection in this region and reveals that these regions are susceptible to evolutionary pressure possibly caused by antibodies what gives a strong verification to our hypothesis that neutralising antibodies are a key determinant in the inherently complex adaptive evolution of HPIV2 in the region.

Antigenic differences between vaccine and circulating wild-type mumps viruses decreases neutralization capacity of vaccine-induced antibodies.

A recent resurgence of mumps in doubly vaccinated cohorts has been observed, identifying genotype G as the current predominant genotype. In this study, the neutralization efficacy of guinea pig sera immunized with three vaccine viruses: L-Zagreb, Urabe AM9 and JL5, was tested against seven mumps viruses: three vaccine strains and four wild-type strains (two of genotype G, one of genotype C, one of genotype D) isolated during 1998-2011. All sera neutralized all viruses although at different levels. The neutralization efficiency of sera decreases several fold by temporal order of virus isolation. Therefore, we concluded that gradual evolution of mumps viruses, rather than belonging to a certain genotype, results in an antigenic divergence from the vaccine strains that decrease the neutralization capacity of vaccine-induced antibodies. Moreover, the amino-acid sequence alignment revealed three new potentially relevant regions for escape from neutralization, i.e. 113-130, 375-403 and 440-443.

Inventory management.

A critical aspect of blood transfusion is the timely provision of high quality blood products. This task remains a significant challenge for many blood services and blood systems reflecting the difficulty of balancing the recruitment of sufficient donors, the optimal utilization of the donor's gift, the increasing safety related restrictions on blood donation, a growing menu of specialized blood products and an ever-growing imperative to increase the efficiency of blood product provision from a cost perspective. As our industry now faces questions about our standard practices including whether or not the age of blood has a negative impact on recipients, it is timely to take a look at our collective inventory management practices. This International Forum represents an effort to get a snap shot of inventory management practices around the world, and to understand the range of different products provided for patients. In addition to sharing current inventory management practices, this Forum is intended to foster an exchange of ideas around where we see our field moving with respect to various issues including specialty products, new technologies, and reducing recipient risk from blood transfusion products.

Factors influencing preclinical in vivo evaluation of mumps vaccine strain immunogenicity.

Immunogenicity testing in animals is a necessary preclinical assay for demonstration of vaccine efficacy the results of which are often the basis for the decision whether to proceed or withdraw the further development of the novel vaccine candidate. However, in vivo assays are rarely, if at all, optimized and validated. Here we clearly demonstrate the importance of in vivo assay (mumps virus immunogenicity testing in guinea pigs) optimization for gaining reliable results and the suitability of Fractional factorial design of experiments (DoE) for such a purpose. By the use of DoE with resolution IV (2IV((4-1))) we clearly revealed that the parameters significantly increasing assay sensitivity were interval between animal immunizations followed by the body weight of experimental animals. The quantity (0 versus 2%) of the stabilizer (fetal bovine serum, FBS) in the sample was shown as non-influencing parameter in DoE setup. However, the separate experiment investigating only the FBS influence, and performed under other parameters optimally set, showed that FBS also influences the results of immunogenicity assay. Such finding indicated that (a) factors with strong influence on the measured outcome can hide the effects of parameters with modest/low influence and (b) the matrix of mumps virus samples to be compared for immunogenicity must be identical for reliable virus immunogenicity comparison. Finally the 3 mumps vaccine strains widely used for decades in the licensed vaccines were for the first time compared in an animal model, and results obtained were in line with their reported immunogenicity in human population supporting the predictive power of the optimized in vivo assay.

Helicobacter pylori and gastric cancer in Croatia.

The seroprevalence of Helicobacter pylori infection was studied in a population from two areas of Croatia with significantly different average gastric cancer (GC) cumulative incidence and mortality rates. In a random sample of 456 blood sera from both areas, which was tested with the ELISA Helicobacter pylori-antibody test, 48.8% of people in the north and 53.3% in the south of the country were found to be infected. The difference between the two areas in the seroprevalence of the infection was not statistically significant, nor did their populations differ in age, sex, educational background, smoking habit or wine consumption. Our results do not point to any association between the prevalence of Helicobacter pylori infection and the level of cumulative incidence and GC mortality levels.

Dynamics of serum hepatitis C virus load and quasispecies complexity during antiviral therapy in patients with chronic hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) infection is a dynamic process during which viral genetic variants continuously develop as a result of the virus adaptation to the host's immune system. The level of viremia and the complexity of the hypervariable region 1 (HVR 1) quasispecies of hepatitis C virus during antiviral therapy reflect the dynamic balance between the viral and host components in response to therapy. OBJECTIVE: The aim of the study was to evaluate the dynamics of HCV viremia and the complexity of the HVR 1 quasispecies during the induction phase of a triple combination therapy regimen in nonresponders to earlier anti-HCV treatment. STUDY DESIGN: Ten patients with chronic hepatitis C undergoing antiviral combination therapy with interferon-alpha, ribavirin, and amantadine were studied. The serum HCV RNA level was monitored by a quantitative RT-PCR assay up to 3 months after start of treatment. The HVR 1 quasispecies complexity was analysed by an "in house" nested RT-PCR mediated single-strand conformation polymorphism (SSCP) assay. RESULTS: Baseline serum HCV RNA levels ranged from 1.94x10(6) to 5.53x10(6) copies/ml. In all patients, HCV subtype 1b was found. At the start of therapy, the SSCP assay revealed a high complexity pattern (at least six SSCP bands) in all patients. None of the patients responded within 4 weeks of treatment, however, the serum HCV RNA level decreased by one to two logs in eight patients. At week 4 after start of treatment, there was a decrease of SSCP bands in five patients. In four patients, SSCP bands remained unchanged and in one patient SSCP bands increased. At month 3 after start of treatment, serum HCV RNA was not detectable in one patient. CONCLUSION: Because of the low number of patients involved in this study, prediction of therapeutical success based on the quasispecies complexity was not possible. Larger studies are urgently needed.

Posttraumatic stress disorder and platelet serotonin measures.

The role of serotonin (5HT) in the pathophysiology of posttraumatic stress disorder (PTSD) has been suggested by the overlap in clinical symptoms between PTSD and psychiatric conditions in which a serotonin dysfunction is implicated, as well as by the therapeutic efficiency of 5HT-related drugs (antidepressants, selective serotonin reuptake inhibitors and monoamine oxidase inhibitors) in alleviating symptoms in PTSD. In the present study, the blood platelet, which has been proposed as a peripheral model for the central serotonergic neurons, has been used to search for alterations in 5HT mechanisms in PTSD. Platelet serotonin level and kinetics of serotonin transporter and monoamine oxidase (MAO-B) were assessed in 63 combat-related PTSD patients and 43 sex and age-matched control subjects. A significant reduction in maximal velocity of platelet MAO-B (approx. 30%), with no changes in the enzyme affinity was observed in our patient sample. Conversely, no alterations in kinetic parameters (V(max), K(m)) of platelet serotonin transporter, as well as in platelet 5HT level, were found in the PTSD group.

Physiological characteristics of platelet/circulatory serotonin: study on a large human population.

The aim of this work was the study of platelet/circulatory serotonin (5-hydroxytryptamine, 5-HT), specifically alternative ways of its measurement and main physiological characteristics. The study was performed on a large human population (N=500) of blood donors of both sexes over the course of a longer time period (17 months). Owing to the heterogeneity in measurement of circulatory serotonin encountered in the literature, three ways of expression were comparatively studied: per unit number of platelets, per unit mass of platelet protein and per unit volume of whole blood. Results demonstrated unimodal distribution of individual frequencies of platelet/circulatory serotonin in the human population with the mean values of 579+/-169 ng 5-HT/10(9) platelets; 332+/-89.9 ng 5-HT/mg protein and 130+/-42.3 ng 5-HT/ml blood (mean+/-S.D.). A progressive decrease of serotonin level with age (18-65 years) was demonstrated, reaching statistical significance between the extreme age groups. No significant differences in the serotonin level between the sexes were observed. No seasonal oscillations in platelet/circulatory serotonin were found. Platelet serotonin demonstrated intra-individual stability over time. Finally, regarding the methodology of measurement, our results demonstrated a good correlation among the above-mentioned ways of expression of platelet/circulatory serotonin. This indicates the possibility of intercomparison of the literature reports expressing this physiological parameter either as 5-HT concentration in platelets or as 5-HT level in the circulation.

[Contribution to the nomenclature of variations of the cystic artery]. / Ein Beitrag zur Nomenklatur der Variationen der Arteria cystica.

INTRODUCTION: Safe procedures for laparoscopic cholecystectomy demand good knowledge of the anatomy of the terminal part of the cystic artery and its variations, and also precise dissection in and around the hepatobiliary triangle. METHOD: Good laparoscopic visualisation enables recognition of the variation of the cystic artery. Our observations are based on 1000 cholecystectomies. RESULTS: We have described and named variations of the terminal part of the cystic artery. Group I comprises the five variations of the cystic artery within the hepatobiliary triangle: (a) "normal" position; (b) frontal cystic artery; (c) backside; (d) multiple; (e) short cystic artery that arises from an aberrant right hepatic artery. Group II consists of variations of the cystic artery that approach--the gallbladder beyond the hepatobiliary triangle: (a) "low-lying"; (b) transhepatic; (c) "recurrent" cystic artery. CONCLUSION: Our classification is simple and easy to memorize and will considerably facilitate safe laparoscopic cholecystectomy.

Gastrointestinal pseudo-obstruction: report of a patient with postoperative pseudo-obstruction.

Postoperative pseudo-obstruction is a rare state of protracted gastrointestinal paresis that may progress to paralysis without the presence of obstructive lesions. Pseudo-obstruction is usually, but not exclusively, associated with an abdominal operative procedure (laparotomy), however, it may occasionally occur following extra-abdominal operations. As differentiated from the usual, 'physiologic'postoperative paresis, pseudo-obstruction persists for more than 7 days. The pathogenesis of postoperative pseudo-obstruction is complex and as yet partially unknown. Whereas the 'physiologic' postoperative gastrointestinal paresis includes short-term functional cholinergic depression of the visceral organs, in pseudo-obstruction focal lesions in the region of Auerbach's plexus, manifesting as visceral neuromyopathy, are involved. That is why the 'physiologic' postoperative paresis never transforms into paralytic ileus, while in pseudo-obstruction such a risk is potentially involved. The treatment for pseudo-obstruction is as a rule conservative. Surgical treatment (cecostomy) is rarely required. Colonoscopic decompresive suction is usually enough to eliminate the risk of colon rupture due to extensive distention by fast growing meteorism. A patient with postoperative pseudo-obstruction is presented.

A contribution to the casuistics of choledochal cysts.

Choledochal cyst is a rare cystic anomaly of bile ducts, primarily affecting the choledochus. The etiology of the disease is unknown, while the symptoms predominated by cholangitis usually occur by the age of thirty. The complications of untreated disease include septic complications, biliary cirrhosis, formation of concrements in cystically dilated bile ducts, and a potential risk of cholangiocarcinoma. Two patients with choledochal cysts type III and IV are presented. The patient with type III choledochal cyst underwent radical treatment, whereas in the patient with type IV choledochal cyst only a palliative procedure could be used. Both patients were men older than 40, and were free from the disease associated sequels for two and five years after the surgery.

Biliary duct reoperations.

Conventional operations for cholelithiasis are rarely associated with postoperative complications. However, when the complications do occur, they are frequently life threatening and require reoperation. These reoperations are associated with a considerably higher risk than primary procedures, and require maximal caution and experience from the surgeon. Reoperations are performed in anatomically altered conditions, and in a patient psychically and physically exhausted and in fear from repeat procedure. Therefore, it is of utmost importance for each primary operation including conventional procedure for cholelithiasis to be carried out at a high professional level, using all technologic achievements available that facilitate and improve the surgeon's work safety. In this way, the need of undesired and hazardous reoperations is minimized. The rate of and indications for reoperation in 530 patients operated on by the conventional procedure for cholelithiasis during the 1994-1999 period are presented.

Alloimmunizations following blood transfusions.

A retrospective study was performed to estimate the rate of alloimmunization against red blood cell (RBC) antigens in hospitalized patients and the frequency and specificity of clinically significant alloantibodies (CSA) detected during routine pretransfusion testing of 10,641 blood samples. Clinically significant alloantibodies were found in 116 out of the 10,641 tested serums (1.09%). The incidence of CSA in two surgical groups of patients was 0.18% and 0.68%, respectively. In patients with hematologic diseases the frequency of CSA was 17.6%, in patients with uremic disease it was 14%, and in patients with cirrhosis hepatis it was 6.9%. The incidence of clinically significant alloantibodies in patients who had one to five exposures to RBC antigens was 5.1%; in patients who had six to fifteen exposures, 87.9%; and in patients with more than fifteen exposures, 6.8%. The most frequently found single alloantibodies were anti-Kell (22%), and the following irregular alloantibodies were found with decreasing frequency: anti-E (20%), anti-D (11%), anti-c (10%), anti-C (7%), anti-Kidd (6%). The most frequent alloantibodies found together with other alloantibodies had anti-Duffy specificity (80%), and the following irregular alloantibodies were found with decreased frequency: anti-C (70%), anti-c (57%), and anti-Kidd (50%).

Acute pancreatitis in the Zabok General Hospital.

Acute pancreatitis is an acute disease of the pancreas due to the organ autodigestion. The disease is still burdened with numerous complications and quite frequently with lethal outcome, in spite of the sophisticated diagnostic and therapeutic methods currently available. The disease has a benign course in a majority of patients (80%), however, in the remaining 20% it assumes a malignant course with the development of massive necroses of the pancreatic and peripancreatic tissues, infection, hemorrhage, and endogenous intoxication with lesions of the lungs, kidneys, heart and liver. The biliary tract disease plays the major role in the etiology of acute pancreatitis (80%), followed by alcoholism (10% to 15%). This differs from the experience acquired at the Zabok General Hospital, where an almost identical incidence of biliary and ethylic etiology was recorded. Other, less common causes include post-traumatic, postoperative, infective and hormonal (hyperparathyroidism) etiology. In some cases, the cause of acute pancreatitis remains unknown. The disease shows a female predominance, which results from the higher prevalence of cholelithiasis in women than in men. Anatomically, there are two main forms of acute pancreatitis, interstitial or edematous form, and hemorrhagic necrotizing form. The interstitial or edematous form of acute pancreatitis is characterized by edema (exudation) of the pancreatic interstitium. The hemorrhagic necrotizing form of acute pancreatitis is characterized by autodigestion of a minor or major portion of the pancreas and peripancreatic tissues. The diagnosis of acute pancreatitis may initially pose a considerable problem. Decision on the mode of treatment should primarily be based on the clinical picture and supported by relevant laboratory parameters and other diagnostic procedures (ultrasonography, computed tomography). Conservative therapy is indicated for the edematous form of acute pancreatitis, whereas operative treatment is as a rule used for the necrotizing form of acute pancreatitis. Secondary bacterial contamination of the necrotic foci with the development of septic complications occurs in more than 50% of patients with the necrotizing form of acute pancreatitis, and is an absolute indication for surgical intervention. The modes of treatment used in 57 patients admitted for acute pancreatitis during the 1996-1999 period are described. Cholelithiasis was the cause of acute pancreatitis in 28 (49.1%), and alcoholism in 29 (50.9%) patients. Conservative treatment was used in 41 (72%) patients. Sixteen (28%) patients underwent operative treatment. Explorative laparotomy and drainage were performed in four patients, and explorative laparotomy, necrectomy, sequestrectomy and drainage with two or more drains in 11 patients. Cholecystectomy and T drainage along with necrectomy and drainage were performed in one patient. There were 12 (21%) patients with the most severe form of acute pancreatitis. Nine of these patients were operated on (necrectomy drainage) between day 6 and 10 of the disease. Two of these patients had to be reoperated on within a month, due to necrosis and abscess recurrence. Three of the 12 patients with the severe form of acute pancreatitis received conservative therapy. Fifteen patients were operated on 8-10 weeks after acute pancreatitis had subsided. Pseudocysts developed in three patients. These patients were operated on 6-8 weeks from the onset of disease, with internal drainage via isolated small intestine flexure performed in all of these patients. The mean duration of intensive care unit stay for all patients with acute pancreatitis was 20.6 days. Four of 57 patients hospitalized for acute pancreatitis died. The mortality rate in the group of patients with the severe form of acute pancreatitis (n = 12) was 33%. Complications developed in 50% of operatively treated patients.

Croatian blood transfusion service in prevention of HIV spread during the war.

Beside the war, an unfavorable epidemiological situation and a large number of foreign peace troops that entered the country without having been previously tested for infectious diseases, the number of AIDS cases in our country remained relatively low. The transfusion service played a considerable part in the prevention of HIV infection spread. Although the blood transfusion service was faced with higher demands for blood and blood products, throughout the period of the war not a single blood unit was imported and no single unit of blood components was transfused without having been previously tested for the presence of viral disease markers. Despite enormous economic difficulties, three new diagnostic tests were then introduced in our transfusion practice as a regular procedure: anti-HCV in 1993, anti-HIV 2 in 1994 and anti-HIV 1/0 in 1995.

Ochratoxin A in blood of healthy population in Zagreb.

Healthy blood donors from the city of Zagreb were checked for the presence of a nephrotoxic mycotoxin ochratoxin A (OTA) in the plasma. Samples of blood were collected in June, September, and December 1997, and March 1998, totalling 200 or 50 in each round. The concentrations of OTA were measured using high pressure liquid chromatography (HPLC) method (detection limit 0.2 ng OTA/ml of plasma). The frequency of OTA-positive samples (> 0.2 ng/ml of plasma) showed significant seasonal variation (P < 0.001). The frequency of OTA-positive samples was the highest in March (65%) and it gradually decreased towards December (12%). The high frequency of positive samples coincided with seasons favouring growth of moulds and production of toxins. The daily intake of OTA by healthy persons in Zagreb was estimated from the mean concentration of OTA in samples collected during the whole year (0.19 ng OTA/ml plasma). The estimated daily intake was 0.26 ng/kg b.w., that is, substantially below the tolerable daily intake proposed by World Health Organization (16.0 ng/kg b.w.).

[Safety of transfusion therapy with special emphasis on inactivated viruses in products made from human blood]. / Sigurnost transfuzijskog lijecenja s posebnim osvrtom na inaktivaciju virusa u preparatima proizvedenim iz ljudske krvi.

The most common complication of blood transfusion therapy is the transfusion-transmitted infection. The decrease of the risk had been achieved by several measures such as: the change from paid to all voluntary donor system, education of the population and potential donor groups as regards the risk factors, and the selection of donors without the risk factors, testing of donor blood for the presence of the hepatitis B and C viruses, HIV and syphilis, fractionation of plasma with the partition of viruses and their inactivation. In spite of all these efforts zero risk blood transfusion therapy has not been achieved and it is very unlikely that it will be achieved in the future. Clinicians must have sufficient background information on different procedures for viral inactivation in order to be able to select the least risky blood product.

[Evaluation of the direct antiglobulin test]. / Evaluacija direktnog antiglobulinskog testa.

The validity of a direct antiglobulin test (DAT) was evaluated by testing 7645 blood samples from hospitalized patients. DAT was routinely done in 7201 blood samples sent for pretransfusion testing and 444 blood samples specifically sent for the examination of immune hemolysis. Positive DAT was discovered in 0.04% (3/7201) pretransfusion samples and in 3.83% (17/444) samples examined for immune hemolysis. In 16 of the samples with positive DAT, IgG antibodies with or without complements and in 4 samples only components of complements were detected on RBC. The cost of positive DAT in pretransfusion testing is 92 times higher than that of DAT during laboratory investigation of immune hemolysis. Due to a low frequency of positive DAT during pretransfusion testing, its cast and the fact that patients had no clinical signs of immune hemolysis, we advocate no use of a routine DAT during pretransfusion testing.

[Quality control in an immunohematology laboratory in the Croatian Transfusion Service]. / Kontrola kvalitete rada u imunohematoloskim laboratorijima transfuzijskih ustanova u hrvatskoj.

In order to evaluate the testing proficiency in immunohematological laboratories in Croatia blood samples were prepared and fully examined in the Croatian Institute for Transfusion Medicine and sent to all the transfusion laboratories in the country. The laboratories were asked to perform the following tests: determination of AB0 blood group and Rh phenotype; detection and identification of irregular antibodies and crossmatches between serum and RBCs. All the laboratories (100%) accurately determined AB0 and Rh(D) negative blood groups and crossmatch between compatible serum and RBCs. In 80.65% of the laboratories, Rh(Du) blood group was accurately determined. The incompatibility between serum and Rh(Du) RBCs in crossmatch was detected in 93.55% of the laboratories. 96.77% laboratories correctly detected irregular antibodies. Only 35.48% of the laboratories accurately identified anti-D and anti-C alloantibodies in the serum, 32.26% failed to identify one of the two antibodies and 29.03% of the laboratories detected irregular antibodies but did not identify their specificity. Only 35.48% of the laboratories correctly performed all the tasks.