Milorad Pavlovic

Intracardiac thrombosis and fever possibly triggered by ovulation induction in a patient with antiphospholipid antibodies

We report on a 28-year old patient with polycystic ovary syndrome (PCOS) who presented with fever and laboratory markers of inflammation. Her medical history was relevant for multiple ovulation inductions (OI) and ovarian hyperstimulation syndrome (OHSS). She had two miscarriages and one preterm delivery. Intracardiac thrombosis was diagnosed in the presence of antiphospholipid antibodies. We suggest that primary antiphospholipid syndrome (APS) was possibly triggered by OI.

Imported malaria in Belgrade, Serbia, between 2001 and 2009

ZusammenfassungSeit 2000 steigt die Zahl serbischer Bürger, die Reisen in die Tropen machen, langsam aber ständig. Um die epidemiologischen und klinischen Charakteristika der nach Serbien verschleppten Malaria zu erfassen, analysierten wir die klinische Anamnese aller Reisenden, die sich nach der Rückkehr aus tropischen oder subtropischen Gebieten zwischen 2001 und 2009 am Institut für Infektionen und Tropenkrankheiten in Belgrad vorstellten. Insgesamt wurden 2981 Reisende erfasst, von denen 847 gesundheitliche Probleme hatten. Eine Malaria wurde bei 102 (3,4 % aller Reisenden; 12 % aller Reisenden mit Fieberschüben) diagnostiziert. Mit einer Inzidenzrate von 6-16 Fälle pro Jahr wurde die Malaria hauptsächlich aus Afrika (92,2 %), vor allem Äquatorial Guinea (38,2 %) und Nigeria (15,7 %) eingeschleppt. Der häufigste Grund für die Reisen war geschäftlich, beziehungsweise beruflich. Die Patienten waren zumeist Männer (87,3 %) in einem Alter großteils zwischen 40 und 59 Jahren (66,7 %). Insgesamt nahmen 15 der erkrankten Personen (14,7 %) irgendeine Form einer Malariaprophylaxe ein. Plasmodium (P) falciparum (78), alleine (70) oder gemischt mit P. vivax (5) war die häufigste ursächliche Spezies. P. vivax, P. ovale und P. malariae konnten in 11, 1 und 1 der Fälle als alleiniger Erreger nachgewiesen werden. Von den 11 Fällen, in denen der Parasit nicht gefunden wurde, schienen sechs echte submikroskopische Fälle zu sein. Der klinische Verlauf war bei 13 Patienten (alle mit P. falciparum infiziert) schwer. Drei dieser Patienten (2,9 %) verstarben. Screening auf Malaria sollte in Serbien für in endemische Regionen Reisende, die sich mit Fieber präsentieren, (unabhängig davon, ob sie eine Chemoprophylaxe einnehmen oder nicht), verpflichtend sein. Nicht ausreichende Sensitivität der konventionellen Methoden (wie wir sie bei den submikroskopischen Fällen beobachtet haben) erfordert die Einführung molekularer Diagnostik in die Routinepraxis.SummarySince 2000, travel of Serbian citizens to tropical areas has been slowly but steadily increasing. To determine the epidemiological and clinical characteristics of imported malaria in Serbia, we analyzed clinical history data of all travelers who presented at the Clinic for Infectious and Tropical Diseases in Belgrade after their return from tropical and subtropical areas between 2001 and 2009. The study series involved a total of 2981 travelers, and included both those with (847) and without (2134) health problems. Malaria was diagnosed in 102 cases (3.4% of all travelers; 12.0% of travelers with febrile episodes). Occurring at a rate of 6 to 16 cases per year, it was predominantly imported from Africa (92.2%), particularly from Equatorial Guinea (38.2%) and Nigeria (15.7%). The most frequent reason for travel was work/business. Patients were predominantly (87.3%) male, and the majority (66.7%) was between 40 and 59 years of age. A total of 15 (14.7%) patients took some form of anti-malarial chemoprophylaxis. The dominant causative species was Plasmodium falciparum (78), alone (70) or in mixed infection with P. vivax (5) and P. malariae (3). P. vivax, P. ovale and P. malariae as single agents were each identified in 11, 1 and 1 cases, respectively. Of the 11 cases in which the parasite was not detected, six appeared to be true submicroscopic cases. The clinical course of the disease was severe in 13 patients, all with falciparum malaria, of which three (2.9%) died. Rather than for all travelers, in Serbia screening for malaria should be mandatory in all travelers to endemic regions who present with fever irrespective of chemoprophylaxis history. Inadequate sensitivity of conventional diagnostic methods, illustrated by the cases of submicroscopic malaria, requires introduction of molecular diagnosis in routine practice.

Mitral valve endocarditis caused by Pseudomonas aeruginosa: a case report

Previously, endocarditis caused by Pseudomonas aeruginosa mostly involved right-sided valvular infection and generally carried a good prognosis. Recently, changes have been observed in the occurrence and clinical presentation of pseudomonal endocarditis, with increasing incidence of nosocomial infections and involvement of the aortic and mitral valves. Still, pseudomonal left-sided endocarditis is rare, but is frequently associated with complications and high mortality rates. A case of a high-risk patient with coronary artery disease and left-ventricular dysfunction, successfully treated for pseudomonal mitral valve endocarditis complicated by splenic abscess formation, is presented here.

Human case of fasciolosis in Serbia treated with triclabendazole

INTRODUCTION The number of humans infected by Fasciola hepatica is increasing worldwide. Humans can become accidental hosts by ingesting drinking water or plants contaminated with metacercariae. CASE REPORT We reported a case of a 68-year-old Serbian woman, in which the diagnosis of acute fasciolosis had been established after serious diagnostic concerns. Based on clinical picture (episodic right upper quadrant abdominal pain, febrility and generalized body pain) and biochemical analyses (high eosinophilia and high activity of alkaline phosphatase), she was appointed as suspected to the acute fasciolosis. Stool and duodenal aspirate exams were negative for Fasciola ova. In the absence of adequate serologic diagnostic for fasciolosis in Serbia, the diagnosis was confirmed using enzyme immunoassays and immunoblot at the Institute for Tropical Diseases in Hamburg, Germany. Soon after triclabendazole was administered, the symptoms disappeared and biochemical values returned to normal. CONCLUSION The diagnosis of human fasciolosis may be problematic and delayed, especially in non endemic areas, because physicians rarely encounter this disease and a long list of other diseases must be considered in the differential diagnosis. The syndrome of eosinophilia, fever, and right upper quadrant abdominal pain suggest acute fasciolosis. Unclear source does not rule out fasciolosis.

Clinical significance of molecular methods in the diagnosis of imported malaria in returning travelers in Serbia

OBJECTIVES The goal of this study was to assess the clinical significance of conventional and PCR-based molecular diagnosis in patients with imported malaria in Serbia. METHODS Giemsa microscopy, the rapid diagnostic test, and quantitative real-time PCR (qPCR) were used to detect Plasmodium species in 109 whole-blood samples from patients after their return from malaria endemic areas, including those clinically suspected for malaria (n=97) and healthy travelers (n=12) examined as part of epidemiological surveillance. RESULTS A total of 45 patients were diagnosed with malaria: 42 (93.3%) by microscopy and three (6.7%) additional ones by qPCR. The agreement between the results of species-specific qPCR and microscopy was 73.3%; it was as high as 90.6% for Plasmodium falciparum infections. Follow-up analysis demonstrated persistence of Plasmodium sp DNA for a mean 6 days after the disappearance of parasitemia on microscopy. CONCLUSIONS Due to its sensitivity and specificity, qPCR is a helpful method complementary to microscopy, particularly in cases of low parasitemia. In addition, it is superior to microscopy for species identification.

Imported parasitic infections in Serbia

BACKGROUND Travel to the tropics is associated with a risk of parasitic infection, which is increasing in parallel with the rise in travel to these areas. We thus examined the prevalence and trend in the occurrence of parasitic infections in Serbian travelers. METHODS A retrospective analysis of the medical records of all travelers returning from tropical and subtropical areas, who presented at the Institute for Infectious and Tropical Diseases in Belgrade between January 2001 and January 2008, was performed. RESULTS Of a total of 2440 travelers, 169 (6.9%) were diagnosed with a parasitic infection, including malaria in 79, intestinal parasites in 84 (pathogenic species in 30 and non-pathogenic in 54), filariasis in four, and visceral leishmaniasis and fascioliasis in one patient each. Importantly, of the whole series only 583 (23.9%) were symptomatic, of which 19.4% were found to be infected with a parasite. The single pathogenic parasite occurring in asymptomatic patients was Giardia intestinalis. CONCLUSIONS Parasitic infection causing symptomatic disease among travelers returning from tropical areas to Serbia is not infrequent. In view of the expected increase in travel to the tropics, diagnostic protocols for tropical parasitic diseases should take these data into account.

Adrenal cortex function impairment in chronic fatigue syndrome

Chronic fatigue syndrome (CFS) is defined as constellation of the prolonged fatigue and several somatic symptoms, in the absence of organic or severe psychiatric disease. However, this is an operational definition and conclusive biomedical explanation remains elusive. Similarities between the signs and symptoms of CFS and adrenal insufficiency prompted the research of the hypothalamo-pituitary-adrenal axis (HPA) derangement in the pathogenesis of the CFS. Early studies showed mild glucocorticoid deficiency, probably of central origin that was compensated by enhanced adrenal sensitivity to ACTH. Further studies showed reduced ACTH response to vasopressin infusion. The response to CRH was either blunted or unchanged. Cortisol response to insulin induced hypoglycaemia was same as in the control subjects while ACTH response was reported to be same or enhanced. However, results of direct stimulation of the adrenal cortex using ACTH were conflicting. Cortisol and DHEA responses were found to be the same or reduced compared to control subjects. Scott et all found that maximal cortisol increment from baseline is significantly lower in CFS subjects. The same group also found small adrenal glands in some CFS subjects. These varied and inconsistent results could be explained by the heterogeneous study population due to multifactorial causes of the disease and by methodological differences. The aim of our study was to assess cortisol response to low dose (1 µg) ACTH using previously validated methodology. We compared cortisol response in the CFS subjects with the response in control and in subjects with suppressed HPA axis due to prolonged corticosteroid use. Cortisol responses were analyzed in three subject groups: control (C) secondary adrenal insufficiency (AI), and in CFS. The C group consisted of 39 subjects, AI group of 22, and CFS group of nine subjects. Subject data are presented in table 1. Low dose ACTH test was started at 0800 h with the iv injection of 1 µg ACTH (Galenika, Belgrade, Serbia). Blood samples for cortisol determination were taken from the iv cannula at 0,15, 30, and 60 min. Data are presented as mean standard error (SE). Statistical analysis was done using ANOVA with the Games-Howell post-hoc test to determine group differences. ACTH dose per kg or per square meter of body surface was not different between the groups. Baseline cortisol was not different between the groups. However, cortisol concentrations after 15 and 30 minutes were significantly higher in the C group than in the AI group. Cortisol concentration in the CFS group was not significantly different from any other group (Graph 1). Cortisol increment at 15 and 30 minutes from basal value was significantly higher in C group than in other two groups. However there was no significant difference in cortisol increment between the AI and CFS groups at any time of the test. On the contrary, maximal cortisol increment was not different between CFS and other two groups, although it was significantly higher in C group than in the AI group. Maximal cortisol response to the ACTH stimulation and area under the cortisol response curve was significantly larger in C group compared to AI group, but there was no difference between CFS and other two groups. Several previous studies assessed cortisol response to ACTH stimulation. Hudson and Cleare analysed cortisol response to 1 µg ACTH in CFS and control subjects.They compared maximum cortisol attained during the test, maximum cortisol increment, and area under the cortisol response curve.There was no difference between the groups in any of the analysed parameters. However, authors commented that responses were generally low. On the contrary Scott et all found that cortisol increment at 30 min is significantly lower in the CFS than in the control group. Taking into account our data it seems that the differences found in previous studies papers are caused by the methodological differences. We have shown that cortisol increment at 15 and 30 min is significantly lower in CFS group than in C group. Nevertheless, maximum cortisol attained during the test, maximum cortisol increment, and area under the cortisol response curve were not different between the C and CFS groups. This is in agreement with our previous findings that cortisol increment at 15 minutes has the best diagnostic value of all parameters obtained during of low dose ACTH test. However, there was no difference between CFS and AI group in any of the parameters, although AI group had significantly lower cortisol concentrations at 15 and 30 minutes, maximal cortisol response, area under the cortisol curve, maximal cortisol increment, and maximal cortisol change velocity than C group. Consequently reduced adrenal responsiveness to ACTH exists in CFS. In conclusion, we find that regarding the adrenal response to ACTH stimulation CFS subjects present heterogeneous group. In some subjects cortisol response is preserved, while in the others it is similar to one found in secondary adrenal insufficiency.

Clinical characteristics of imported malaria: An 11-year experience in a Serbian referral center

INTRODUCTION Due to intercontinental traffic, population migration trends, natural disasters, and climate change, imported malaria remains important to consider in a febrile returning traveler. This study aims to raise awareness about malaria and help European clinicians maintain a working knowledge of this disease by reviewing the most important clinical characteristics in a non-endemic setting. METHODOLOGY Using medical records, a retrospective study was performed on clinical and laboratory data in order to analyze 103 malaria cases managed at the Clinic for Infectious and Tropical Diseases in Belgrade, from 2000 to 2010. Descriptive statistics, Chi-squared test, Spearmans rank correlation, and analysis of variance were used. RESULTS Patients were predominantly male (89.3%) with a mean age of 46.66 ± 12.45 years, and most (98.06%) returned from Africa without having taken chemoprophylaxis (72.88%). Fever, arthralgia, myalgia, headache, vomiting, dark urine, and cough were common at presentation. Hepatosplenomegaly, jaundice, neurological and pulmonary findings, and thrombocytopenia were dominant findings on physical and laboratory examinations. Most (73.48%) were infected with P. falciparum. Few patients (17.55%) who were hyperparasitemic had significantly higher values of bilirubin and more frequent neurological complications. All patients were treated with artemisinin-based drug combinations regardless of Plasmodium species. Three (2.9%) patients succumbed to P. falciparum malaria. CONCLUSION We suggest a high index of suspicion of malaria be maintained when evaluating febrile patients returning from endemic regions, especially if thrombocytopenia and hemolysis are present. Hyperparasitemia, high bilirubin levels, and neurological symptoms are associated with severe malaria. The importance of adequate malaria chemoprophylaxis cannot be overstated.

Retrospective PCR-based species identification of Leishmania in two patients with visceral leishmaniasis in Serbia

Introduction: Retrospective molecular identification of Leishmania parasites in two patients with visceral leishmaniasis (VL) previously treated in Serbia was carried out. DNA was isolated from unstained bone marrow smears (BMSs) kept for 11 and 8 years. Genus-specific real-time PCR was combined with conventional PCR and sequencing for detection and species identification. Case presentation: In 2003, a 40-year-old Serbian male was admitted to the Clinical Centre of Serbia (CCS) with fever, sweating, fatigue and splenomegaly, which developed over a period of 7 weeks. He had frequently travelled around Europe. VL was confirmed by microscopy of Giemsa-stained BMS. Treatment by pentavalent antimonials was successfully completed. Two years later, the patient developed post-kala-azar dermal leishmaniasis. Treatment resulted in symptom resolution. Later on, Leishmania infantum was identified as the causative agent of the VL by sequencing of the ITS (internal transcribed spacer) region; mixed Leishmania spp. infection could not be excluded. In 2006, a 33-year-old female from Vojvodina, Serbia, with pre-existing diabetes mellitus and chronic meningoencephalitis and a history of frequent visits to the Montenegrin seacoast, was admitted to the CCS with fever, pancytopenia and moderate hepatosplenomegaly. A stained BMS revealed abundant Leishmania amastigotes. Indirect haemagglutination analysis was positive with a titre of 1 : 2048, and a rapid dipstick rK39 test was also positive. Treatment by liposomal amphotericin B was successful; however, shortly after, the patient developed neural infection and pneumonia and died. The causative agent was identified as L. infantum. Conclusion: Molecular diagnosis of VL and species delineation using DNA from unstained BMSs stored for several years is possible.

Size of the protein-coding genome and rate of molecular evolution

AbstractIn diploid populations of size N, there will be 2 Nμ mutations per nucleotide (nt) site (or per locus) per generation (μ stands for mutation rate). If either the population or the coding genome double in size, one expects 4 Nμ mutations. What is important is not the population size per se but the number of genes (coding sites), the two being often interconverted. Here we compared the total physical length of protein-coding genomes (n) with the corresponding absolute rates of synonymous substitution (KS), an empirical neutral reference. In the classical occupancy problem and in the coupons collector (CC) problem, n was expressed as the mean rate of change (KCC). Despite inherently very low power of the approaches involving averaging of rates, the mode of molecular evolution of the total size phenotype of the coding genome could be evidenced through differences between the genomic estimates of KCC [KCC=1/(ln n + 0.57721) n] and rate of molecular evolution, KS. We found that (1) the estimates of n and KS are reciprocally correlated across taxa (r=0.812; p≪ 0.001); (2) the gamete-cell division hypothesis (Chang et al. Proc Natl Acad Sci USA 91:827-831, 1994) can be confirmed independently in terms of KCC/KS ratios; (3) the time scale of molecular evolution changes with change in mutation rate, as previously shown by Takahata (Proc Natl Acad Sci USA 87:2419-2423, 1990), Takahata et al. (Genetics 130:925-938, 1992), and Vekemans and Slatkin (Genetics 137:1157-1165, 1994); (4) the generation time and population size (Lynch and Conery, Science 302:1401-1404, 2003) effects left their “signatures” at the level of the size phenotype of the protein-coding genome.