The importance of minimal residual disease for detection of late relapse in Bprecursor acute lymphoblastic leukemia.

BACKGROUND: The minimal residual disease (MRD) level in patients with B-precursor acute lymphoblastic leukemia (B-ALL) is the strongest independent predictor of relapse and survival. Assessment of MRD plays a crucial role in the treatment of B-ALL. CASE REPORT: We performed long-term monitoring of a 30-year-old woman with B-ALL of standard risk for MRD using multiparametric flow cytometry (MFC). After five years of monitoring, molecular relapse of the disease was confirmed. CONCLUSION: This case illustrates that more extended monitoring for MRD, even by only MFC when other newer sophisticated diagnostics are not available, is essential in detecting early relapse in patients with B-ALL of standard risk. HIPPOKRATIA 2022, 26 (1):38-40.

Voxel model of a rabbit: assessment of absorbed doses in organs after CT examination performed by two different protocols.

The objective of this work was to assess absorbed doses in organs and tissues of a rabbit, following computed tomography (CT) examinations, using a dedicated 3D voxel model. Absorbed doses in relevant organs were calculated using the MCNP5 Monte Carlo software. Calculations were perfomed for two standard CT protocols, using tube voltages of 110 kVp and 130 kVp. Absorbed doses were calculated in 11 organs and tissues, i.e., skin, bones, brain, muscles, heart, lungs, liver, spleen, kidney, testicles, and fat tissue. The doses ranged from 15.3 to 28.3 mGy, and from 40.2 to 74.3 mGy, in the two investigated protocols. The organs that received the highest dose were bones and kidneys. In contrast, brain and spleen were organs that received the smallest doses. Doses in organs which are stretched along the body did not change significantly with distance. On the other hand, doses in organs which are localized in the body showed maximums and minimums. Using the voxel model, it is possible to calculate the dose distribution in the rabbit's body after CT scans, and study the potential biological effects of CT doses in certain organs. The voxel model presented in this work can be used to calculated doses in all radiation experiments in which rabbits are used as experimental animals.

Resolving the taxonomic status of biocontrol agents belonging to the Aphidius eadyi species group (Hymenoptera: Braconidae: Aphidiinae): an integrative approach.

Species that belong to the Aphidius eadyi group have been used as biocontrol agents against Acyrthosiphon pisum worldwide. However, despite their extensive use, there are still gaps in our knowledge about their taxonomy and distribution. In this study, we employed an integrative taxonomic approach by combining genetic analyses (mtDNA COI barcoding) with standard morphological analyses and geometric morphometrics of forewing shape. We identified three species within the A. eadyi species group, viz., A. smithi, A. eadyi and A. banksae. Genetic separation of all three species was confirmed, with mean genetic distances between species ranging from 5 to 7.4%. The following morphological characters were determined as the most important for separating species of the A. eadyi group: number and shape of costulae on the anterolateral part of the petiole, shape of the central areola on the propodeum, and shape and venation of the forewings. The differences in wing shape of all three species were statistically significant, but with some overlapping. We identified A. banksae as a widely distributed pea aphid parasitoid, whose known range covers most of the western Palaearctic (from the UK to Israel). Aphidius banksae is diagnosed and redescribed.

DIABETIC NEPHROPATHY IN PREGNANT WOMEN WITH TYPE 1 DIABETES (MULTIPLE CASES REVIEW AND DISCUSSION).

Pregnancy in women with pre-existing diabetes is associated with a doubled, or even up to four-fold increased risk of preeclampsia, preterm delivery, and perinatal mortality compared to the risk observed in a background population. Diabetic nephropathy (DN) is a progressive disease that affects about one third of diabetic patients and is among the most frequent causes of the end stage renal disease (ESRD) worldwide. In the random population, it is present in up to 7% of women with type 1 diabetes mellitus (T1DM), and it represents the most common chronic kidney disease occurring in graviditas, complicating between 2.5% and 5% of T1DM pregnancies. Although maternal and perinatal morbidity and mortality rates in the pregnancies complicated with DN have declined over time, pregnancy related health complications are still more common in women with DN compared to those observed among diabetic women without DN. The adequate pre-pregnancy counseling and a tailored approach to the treatment of women with DN during the pregnancy are crucial for an avoidance of the adverse maternal and fetal outcomes. This paper is highlighting the impact of DN on maternal and fetal outcomes in women with T1DM, through presentation of the cases from a real-world clinical practice.

Genetic differentiation of Liparus glabrirostris (Curculionidae: Molytinae) populations from the fragmented habitats of the Alps and Carpathian Mountains.

Populations of Liparus glabrirostris (Curculionidae: Molytinae), a weevil inhabiting higher altitudes of Central Europe, were sampled from 24 localities in the Alps and Carpathian Mountains, and the geographical structuring of genetic variation was analyzed. Comparison of the concatenated mitochondrial cytochrome oxidase subunit I and subunit II sequences revealed consistent genetic divergence between the populations of L. glabrirostris from different mountain ranges. In phylogenetic analysis using maximum parsimony and median-joining networks, concatenated mitochondrial haplotypes from the Alps and Carpathians clustered as separate lineages, with high bootstrap support. Substantial genetic distances determined between the separated groups ranged from 2.6 to 3.0%, with divergence estimated to have initiated approximately 0.85-0.98 million years ago. The nuclear elongation factor 1α gene was additionally amplified and haplotype analysis showed very low evolutionary divergence (0.2%), with separate clustering as well. The observed divergence suggests that the populations have been isolated for a long time, as a consequence of environmental changes resulting in varying fragmentation of habitats in the Alps and Carpathians, interrupting genetic exchange events and altering the genetic structure of L. glabrirostris populations. On the other hand, comparison of morphological characteristics showed no differences to confirm genetically well differentiated groups of populations. A polymerase chain reaction and restriction fragment length polymorphism-based method was therefore developed to discriminate between the Alpine and Carpathian lineages.

Dictyophara europaea (Hemiptera: Fulgoromorpha: Dictyopharidae): description of immatures, biology and host plant associations.

The European lantern fly Dictyophara europaea (Linnaeus, 1767), is a polyphagous dictyopharid planthopper of Auchenorrhyncha commonly found throughout the Palaearctic. Despite abundant data on its distribution range and reports on its role in the epidemiology of plant-pathogenic phytoplasmas (Flavescence dorée, FD-C), literature regarding the biology and host plants of this species is scarce. Therefore, the aims of our study were to investigate the seasonal occurrence, host plant associations, oviposition behaviour and immature stages of this widespread planthopper of economic importance. We performed a 3-year field study to observe the spatio-temporal distribution and feeding sources of D. europaea. The insects's reproductive strategy, nymphal molting and behaviour were observed under semi-field cage conditions. Measurement of the nymphal vertex length was used to determine the number of instars, and the combination of these data with body length, number of pronotal rows of sensory pits and body colour pattern enabled the discrimination of each instar. We provide data showing that D. europaea has five instars with one generation per year and that it overwinters in the egg stage. Furthermore, our study confirmed highly polyphagous feeding nature of D. europaea, for all instars and adults, as well as adult horizontal movement during the vegetation growing season to the temporarily preferred feeding plants where they aggregate during dry season. We found D. europaea adult aggregation in late summer on Clematis vitalba L. (Ranunculaceae), a reservoir plant of FD-C phytoplasma strain; however, this appears to be a consequence of forced migration due to drying of herbaceous vegetation rather than to a high preference of C. vitalba as a feeding plant. Detailed oviposition behaviour and a summary of the key discriminatory characteristics of the five instars are provided. Emphasis is placed on the economic importance of D. europaea because of its involvement in epidemiological cycles of phytoplasma-induced plant diseases.

'On-demand' romiplostim therapy in immune thrombocytopenia.

WHAT IS KNOWN AND OBJECTIVE: Although thrombopoietin receptor agonists are a second-line treatment for refractory immune thrombocytopenia (ITP), we lack guidelines recommending maintenance modality in patients who achieve complete remission (CR). CASE SUMMARY: We report a patient with refractory ITP who achieved CR on romiplostim. Obtaining romiplostim for 6 months of therapy, we decided to try extending this by modifying the standard treatment regimen. Romiplostim was successfully administered 'on-demand', only if the patient's platelet count dropped below 150 × 109 /L, over a period of 12 months. WHAT IS NEW AND CONCLUSION: The strategy of 'on-demand' therapy is a promising procedure for the maintenance of response, lowering costs and improving treatment safety.

Assessment of the contamination of riparian soil and vegetation by trace metals--A Danube River case study.

The aim of this study was to assess the spatial distribution of arsenic and heavy metals (Cd, Cr, Cu Hg, Ni, Pb and Zn) in a riparian area influenced by periodical flooding along a considerable stretch of the Danube River. This screening was undertaken on soil and plant samples collected from 43 sites along 2386 km of the river, collected during the international Joint Danube Survey 3 expedition (ICPDR, 2015). In addition, data on the concentration of these elements in river sediment was used in order to describe the relationship between sediment, riparian soil and riparian plants. A significant positive correlation (Spearman r, for p<0.05) was found for trace metal concentrations in river sediment and soil (r=0.817). A significant correlation between soil and plants (r=0.438) and sediment and plants (r=0.412) was also found for trace metal concentrations. Elevated levels of Cd, Cr, Cu, and Ni were found at certain sites along the Serbian stretch, while elevated concentrations of Hg were also detected in Hungary, of Pb along the Romanian stretch and of As along the Bulgarian stretch (the Lower Danube). These results point to the presence of naturally-occurring metals derived from ore deposits in the Danube River Basin and anthropogenic metals, released by mining and processing of metal ores and other industrial facilities, which are responsible for the entry of metals such as Cu, Ni and Zn. Our results also indicated toxic Cd and Zn levels in plant samples, measured at the Hercegsznato site (Middle Danube, Hungary), which highlighted these elements as a potential limiting factor for riparian vegetation in that area. The distribution of the analysed elements in plant material also indicates the species-specific accumulation of trace metals. Based on our results, the Lower and Middle Danube were found to be more polluted in terms of the analysed elements.

The Impact of Generic Substitution on Health and Economic Outcomes: A Systematic Review.

BACKGROUND: Generic drugs are considered therapeutically equivalent to their original counterparts and lower in acquisition costs. However, the overall impact of generic substitution (GS) on global clinical and economic outcomes has not been conclusively evaluated. OBJECTIVE: To test whether (1) generics and original products yield the same health outcomes, and (2) generic therapies save economic resources versus original therapies. METHODS: We performed a systematic literature review in Medline, Embase, and the Cochrane Database of Systematic Reviews to identify original studies that examine clinical or economic outcomes of GS. After standardized data extraction, reported outcomes were categorized as supporting or rejecting the hypotheses. Each reported outcome was assessed and accounted for supporting and opposing GS. One publication could provide multiple outcome comparisons. RESULTS: We included 40 studies across ten therapeutic areas. Fourteen studies examined patients on de novo therapy; 24 studies investigated maintenance drug therapy, and two studies considered both settings. Overall, 119 outcome comparisons were examined. Of 97 clinical outcome comparisons, 67% reported no significant difference between generic drugs and their off-patent counterparts. Of 22 economic comparisons, 64% suggested that GS increased costs. Consequently, hypothesis (1) was supported but hypothesis (2) was not. We found no major differences among studies that investigated clinical outcomes with de novo or maintenance therapy. CONCLUSION: The review suggests that clinical effects are similar after GS. However, economic savings are not guaranteed. More systematic research comparing clinical and economic outcomes with or without GS is needed to inform policy on the use of generic substitution.

Primary melanoma of the vagina: a case report and review of literature.

Summary Primary melanoma of the vagina is a rare and very aggressive tumor with an incidence of only 0.46 per one million women per year and less than 250 cases reported in the literature. Here the authors present a case of a 60-year-old woman, gravida 5, para 5, post-menopausal by 28 years, admitted to the Clinic for Obstetrics and Gynecology, with recurrent vaginal bleeding for the last year and with the complaint of a palpable tumor near the vaginal introitus. The preoperative biopsy revealed melanoma. CT scan did not prove she had distant metastasis. The patient was treated surgically, with wide local excision of four x five cm measured lesion and safety margins of two cm. Bilateral inguinal lymphadenectomy was performed. Follow-up five months after initial diagnosis, revealed no evidence of local recurrency or distant metastasis.

Molecular and morphological variability within the Aphidius colemani group with redescription of Aphidius platensis Brethes (Hymenoptera: Braconidae: Aphidiinae).

We have identified the following three taxa related to the Aphidius colemani species group, which are important biological control agents: Aphidius colemani, Aphidius transcaspicus and Aphidius platensis. Using partial sequences of the mitochondrial cytochrome oxidase subunit I (mtCOI) gene and geometric morphometric analysis of the forewing shape, we have explored the genetic structure and morphological variability of the A. colemani group from different aphid host/plant associations covering a wide distribution area. The topology of the maximum parsimony and maximum likelihood trees were identical with 98-100% bootstrap support, clustering A. colemani, A. platensis and A. transcaspicus into separate species. The distances among the taxa ranged from 2.2 to 4.7%, which is a common rate for the between-species divergence within the subfamily Aphidiinae. Differences in the shape of the forewing investigated within the biotypes of A. colemani group are congruent with their genetic diversification. Both A. platensis and A. colemani share a common host range pattern, and it would be interesting to estimate and compare the role of these two species in future biological control strategies against aphids of economic importance. Our results indicate that 'genetic screening' is a reliable approach for identification within the A. colemani group. The high variation in the wing shape among species, including a significant divergence in the wing shape among specimens that emerged from different hosts, makes the forewing shape and wing venation less reliable for species determination. Aphidius platensis is diagnostified and redescribed, and the key for the A. colemani group is presented.

The G8 screening tool detects relevant geriatric impairments and predicts survival in elderly patients with a haematological malignancy.

The G8 screening tool was developed to separate fit older cancer patients who were able to receive standard treatment from those that should undergo a geriatric assessment to guide tailoring of therapy. We set out to determine the discriminative power and prognostic value of the G8 in older patients with a haematological malignancy. Between September 2009 and May 2013, a multi-dimensional geriatric assessment was performed in consecutive patients aged ≥67 years diagnosed with blood cancer at the Innsbruck University Hospital. The assessment included (instrumental) activities of daily living, cognition, mood, nutritional status, mobility, polypharmacy and social support. In parallel, the G8 was also administered (cut-off ≤ 14). Using a cut-off of ≥2 impaired domains, 70 % of the 108 included patients were considered as having an impaired geriatric assessment while 61 % had an impaired G8. The G8 lacked discriminative power for impairments on full geriatric assessment: sensitivity 69, specificity 79, positive predictive value 89 and negative predictive value 50 %. However, G8 was an independent predictor of mortality within the first year after inclusion (hazard ratio 3.93; 95 % confidence interval 1.67-9.22, p < 0.001). Remarkably, patients with impaired G8 fared poorly, irrespective of treatment choices (p < 0.001). This is the first report on the clinical and prognostic relevance of G8 in elderly patients with haematological malignancies. Although the G8 lacked discriminative power for outcome of multi-dimensional geriatric assessment, this score appears to be a powerful prognosticator and could potentially represent a useful tool in treatment decisions. This novel finding certainly deserves further exploration.

First Report of Alder Yellows Phytoplasma Associated with Common Alder (Alnus glutinosa) in the Republic of Macedonia.

Alder yellows phytoplasma (AldYp) is classified as a member of the 16SrV-group of phytoplasmas and is closely related to Flavescence dorée (FD), a quarantined pathogen of economic importance affecting vineyards across Europe. AldYp is associated with common (Alnus glutinosa) and grey alder (A. incana), and has been reported in France, Italy, Germany, Austria, Switzerland, the Baltic region, Serbia, and Montenegro (1,2,4). For Macedonian vineyards, so far, neither infection of grapevine with 16SrV-group of phytoplasmas nor the presence of the main FD phytoplasma vector, Scaphoideus titanus, has been recorded. However, the presence of FD-related phytoplasma was detected in wild Clematis vitalba. In September and October 2013, leaves with petioles from A. glutinosa exhibiting leaf discoloration and yellowing were collected from two sites (41°23'43″ N, 22°54' E and 41°23' N, 22°53' E) in southeast Macedonia near the village of Smolare (Strumica district). Eight samples were collected from each site. Leaves of six asymptomatic alder seedlings collected from the same sites served as a control. Nucleic acids were extracted from fresh leaf midribs and petioles using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Initial phytoplasma identification was carried out by nested PCR assay of the 16S rRNA gene, using universal primers P1/P7 and R16F2n/R16R2 followed by RFLP with MseI endonuclease (Fermentas, Vilnius, Lithuania), as previously reported (4). Characterization of detected phytoplasmas was performed by amplifying two genetic loci specific for the members of the 16SrV group phytoplasmas; the ribosomal protein gene operon (rp) using primers rp(V)F1/rpR1 and rp(V)F1A/rp(V)R1A (3), and the non-ribosomal metionine aminopeptidase (map) gene using primer set FD9f5/MAPr1 and FD9f6/MAPr2 (1). The PCR amplicons were sequenced and deposited in NCBI GenBank database under the accession numbers KJ605448 to 52 (map) and KJ605453 to 57 (rp). The obtained sequences were compared with reference sequences of the 16SrV-group phytoplasmas (1,3) using the neighbor-joining method in MEGA5 (5). The presence of phytoplasma was detected in 14 of 16 symptomatic alder samples, while all control plants tested negative. The MseI restriction profiles were identical among all 14 samples and with the reference strains of the 16SrV group phytoplasmas (EY1 - 16SrV-A, FD-C - 16SrV-C, and FD-D - 16SrV-D). The rp-based phylogeny enabled identification of four diverse phytoplasma strains among the AldYp strains from Macedonia. Three strains clustered within the rpV-E subgroup while one belonged to rpV-L subgroup. Phylogenetic analysis of the more variable genetic locus, map, showed the presence of five diverse phytoplasma strains. Four strains belonged to the map-FD2 (FD-D, FD92) cluster, while one grouped within the map-FD1 (FD70) cluster. To our knowledge, this is the first report of 16SrV phytoplasma group occurrence on alder in Macedonia. The significant similarity between AldYp strains and FD sensu stricto indicate the risk of pathogen exchange between the wild ecosystem and the grapevine (1). Alder trees naturally infected with the FDp-related strains could therefore represent a serious risk for FD outbreak in Macedonian vineyards if local S. titanus populations developed. References: (1) G. Arnaud et al. Appl. Environ. Microbiol. 73:4001, 2007. (2) T. Cvrkovic et al. Plant Pathol. 57:773, 2008. (3) M. Martini et al. Int. J. Syst. Evol. Microbiol. 57:2037, 2007. (4) S. Radonjic et al. Plant Dis. 97:686, 2013. (5) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

First Report of Cercospora apii, Causal Agent of Cercospora Early Blight of Celery, in Serbia.

Celery (Apium graveolens var. dulce) is a very important vegetable crop intensively cultivated in eastern and southern Serbia. During a field survey in August and September 2012, we observed symptoms similar to those of Cercospora early blight in eastern Serbia, with some of the affected fields showing up to 80% disease severity. The lesions on leaves were amphigenous, subcircular to angular and more or less confluent. Lesions enlarged and merged with age, followed by the development of necrotic area causing a continuous deterioration of the plant. Conidiophores arising from the stromata formed dense fascicles, sometimes appearing solitary, brown at the base, paler toward the apex, simple, straight to slightly curved, and rarely geniculate (dimensions 40 to 90 × 5 to 8 µm). Conidia were solitary, hyaline, at first cylindro-obclavate then acicular to acicular-obclavate, straight to slightly curved, subacute to obtuse at the apex, while truncated and thickened at the base (dimensions 45 to 160 × 4 to 5 µm), 5 to 13 septate. Based on the morphological features, we identified the pathogen as Cercospora apii Fresen. (2). In order to obtain monosporic isolates of the fungus, single conidia were cultivated on potato dextrose agar (PDA). To confirm the pathogenicity of the isolates, 5 mm-diameter mycelial plugs from the PDA plates were placed upside down on the adaxial leaf surface of 2-week-old celery seedlings of cv. Yuta. Control plants were inoculated with a sterile PDA plug. Three leaves per plant were disinfected with 70% ethanol, epidermis was scratched with a sterile needle to promote the infection, and inoculated. A total of 12 plants were inoculated with the mycelial plugs and 12 were used as control plants. Inoculated and control plants were kept in a moist chamber for 48 h and then transferred to a greenhouse at 25 ± 2°C. After 2 weeks, the first necrotic spots appeared on inoculated leaves, similar to the symptoms manifested in the field, while control plants remained symptomless. The pathogen was re-isolated and its identity was verified based on morphological and molecular features. To confirm the pathogen's identity, three isolates (CAC4-1, CAC24, and CAC30) were subjected to molecular identification based on the internal transcribed spacer region (ITS) using the ITS1/ITS4 universal primers (5), a partial calmodulin gene (CAL) using CAL-228F/CAL2Rd primers (1,4), and partial histone H3 gene (H3) using CYLH3F/CYLH3R primers (3). Sequences of the amplified regions were deposited in GenBank under accessions KJ210596 to KJ210604. The BLAST analyses of the ITS sequences revealed 100% identity with several Cercospora species (e.g., C. apii [JX143532], C. beticola [JX143556], and C. zebrina [KC172066]), while sequences of CAL and H3 showed 100% identity solely with sequences of C. apii (JX142794 and JX142548). Based on combined morphological and molecular data, the pathogen infecting celery was identified as C. apii, which to our knowledge represents the first report of the presence of the causal agent of Cercospora early blight disease in Serbia. References: (1) I. Carbone and L.M. Kohn. Mycologia 91:553, 1999. (2) P. W. Crous and U. Braun. CBS Biodivers. Ser. 1:1, 2003. (3) P. W. Crous et al. Stud. Mycol. 50:415, 2004. (4) J. Z. Groenewald. Stud. Mycol. 75:115, 2013. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

First Report of Cercospora carotae, Causal Agent of Cercospora Leaf Spot of Carrot, in Serbia.

Carrot (Daucus carota L. subsp. sativus [Hoffm.] Arcang.) is an important vegetable in Serbia, where it is grown on nearly 8,000 ha. In August 2012, ~1,500 ha of carrot fields were inspected in southern Backa in North Serbia. In nearly 40% of the fields, severe foliar and stem symptoms characteristic of cercospora leaf spot of carrot, caused by Cercospora carotae (Pass.) Solheim (3), were observed. Lesions on stems were oblong, elliptical, and more or less sunken, while those on the leaves were amphigenous, subcircular, light brown in the center, and surrounded by a dark brown margin. Conidiophores emerging from the lesions formed very loose tufts but sometimes were solitary. Conidiophores were simple and straight to subflexuous with a bulbous base (17 to 37 × 3 to 5 µm). Conidia were 58 to 102 × 2 to 4 µm, solitary, cylindrical to narrowly-obclavate, and hyaline to subhyaline with 2 to 6 septa. To obtain monosporial isolates, the conidia from one lesion were placed on water agar plates at 25°C in the dark for 24 h, after which single germinated conidia were selected and each placed on a petri dish containing potato dextrose agar (PDA). To confirm pathogenicity of three of the isolates, Koch's postulates were tested on carrot seedlings (3-true-leaf stage of growth) of a Nantes cultivar, SP-80, with 12 plants tested/isolate and 12 non-inoculated plants used as a control treatment. The leaves were atomized until runoff with the appropriate C. carotae spore suspension (104 conidia/ml sterilized water), while control plants were atomized with sterile water. All plants were then incubated in a dew chamber for 72 h, then transferred to a greenhouse at 25 ± 2°C. After 2 weeks, characteristic symptoms resembling those observed in the field developed on all inoculated plants; control plants were asymptomatic. The pathogen was re-isolated from all inoculated plants, and identity of the re-isolated fungi confirmed morphologically as described above, and molecularly as described below. The pathogenicity test was repeated with no significant differences in shape and size of lesions, or dimensions of conidiophores and conidia among isolates. To verify the pathogen identity molecularly, the 28S rDNA was amplified and sequenced using the V9G/LR5 primer set (2,4) as well as internal primers OR-A (5'-ATACCCGCTGAACTTAAGC-3') and 2R-C (5'-AAGTACTTTGGAAAGAG-3'); the ITS region of rDNA using the ITS1/ITS4 universal primers (5); and histone H3 gene (H3) using the CylH3F/CylH3R primers (1). The sequences for the three isolates were deposited in GenBank as Accession Numbers KF468808 to KF468810, KF941306 to KF941308, and KF941303 to KF941305 for the 28S rDNA, ITS and H3 regions, respectively. BLAST results for the ITS sequences indicated 94% similarity to the ITS sequence of an isolate of Pseudocercosporella capsellae (GU214662) and 92% similarity to the ITS sequence of an isolate of C. capsici (HQ700354). The H3 sequences shared 91% similarity with that of several Cercospora spp., e.g., C. apii (JX142548), C. beticola (AY752258), and C. capsici (JX142584), all of which shared the same amino acid sequence of the encoded H3 protein. Also, the 28S rDNA sequences had 99% similarity (identity of 318/319, with 0 gaps) with the single sequence of C. carotae available in GenBank (AY152628), which originated from Norway. This is, to our knowledge, the first report of C. carotae on carrot crops in Serbia as well as southeastern Europe. References: (1) P. W. Crous et al. Stud. Mycol. 50:415, 2004. (2) G. S. de Hoog and A. H. G. Gerrits van den Ende. Mycoses 41:183, 1998. (3) W. G. Solheim. Morphological studies of the genus Cercospora. University of Illinois, 1929. (4) R. Vilgalys and M. Hester. J. Bacteriol. 172:238, 1990. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

Uterine tumor resembling ovarian sex cord tumor. Case report and review of literature.

A uterine tumor resembling an ovarian sex cord tumor (UTROSCT) shows a poly phenotypic immunophenotype with coexpression of epithelial, myoid, and sex cord markers, as well as hormone receptors. The authors present a case of a 59-year-old multiparous woman admitted to the Institute of Gynecology and Obstetrics Clinical Centre of Serbia in January 2010 due to prolonged vaginal bleeding and abdominal discomfort. The vaginal ultrasound showed an enlarged uterus size of 100 x 74 x 81 mm, with extended cavity with an unhomogenic content and myomas sized 54 x 69 mm located in fundus with secondary changes. She underwent abdominal hysterectomy with adnexectomy. Microscopic examination revealed submucosal uterine tumor with variabile histological organization that had anastomotic trabeculae with solid cellular grupations. Rare mitotic figures (2/10 HPF) were found. Additional imunohistochemistry showed immunophenotype: the sex cord areas were positive for vimentin(++), aSMA(++), AE1/AE3(+), PR(+), and ER(+). The poly phenotypic immunophenotype can be useful in differential diagnosis from other neoplasms but also suggests an origin of UTROSCT from uncommitted stem cell enabling for multidirectional differentiation.

Perinatal outcome of singleton pregnancies following in vitro fertilization.

PURPOSE OF INVESTIGATION: To determine whether in vitro fertilization/intracytoplasmatic sperm injection (IVF/ICSI) singleton pregnancies are at increased risk for maternal and fetal complications than spontaneous singleton conceptions. MATERIALS AND METHODS: The pregnancy outcome of 634 singleton pregnancies after IVF/ICSI delivered at the Clinic for Gynecology and Obstetrics during the period January 2006 to January 2010 were compared to 634 matched singleton controls, matched one by one by age, parity, education, and body mass index (BMI). Differences in pregnancy outcomes between the groups were assessed using Student's t-test with Yates correction for continuous variables and Chi-squared test for categorical variables. RESULTS: The mean gestational age at delivery of the IVF group was 38.13 +/- 1.72 weeks, slightly shorter than spontaneously conceived singletons at 38.65 +/- 1.79 weeks. The diagnosis of gestational diabetes mellitus (GDM) was frequently made in the IVF group (11.82% vs 8.35%, t = 2.052, p < 0.05). Total preterm delivery rate of IVF pregnancies was 9.30%, significantly higher than the controls 5.85% (t = 2.33, p < 0.05), especially at the 30-32 weeks gestation period. The predominant mode of delivery after IVF pregnancy was cesarean section (80.75% vs 31.38% at spontaneously conceived, t = 17.71, p < 0.001), while vaginal route was the choice for naturally originated pregnancies 68.6% vs 19.24% (p < 0.01). No differences were found in the average birth weights, LBW, VLBW, SGA, and LGA regarding the pregnancy origin. Perinatal mortality rates were comparable among singletons with different pregnancy origin. CONCLUSIONS: Singletons from IVF/ICSI pregnancies have poorer perinatal outcome associated with higher rates of cesarean sections, preterm birth and prematurity, fetal malpresentation (breech presentation), and the occurrence of maternal GDM in pregnancy.

First Report of Alder Yellows Phytoplasma Infecting Common and Grey Alder (Alnus glutinosa and A. incana) in Montenegro.

Alder yellows phytoplasmas (AldYp) of the 16SrV-group associated with common alder (Alnus glutinosa) and grey alder (A. incana) are closely related to the grapevine yellows (GY)-associated quarantine phytoplasma Flavescence dorée (FDp). AldYp have been reported in several countries where epidemic appearance of FDp has been confirmed (France, Italy, and Serbia) (1,2). To date, the presence of 16SrV-group of phytoplasmas has not been reported in Montenegro; however, the main vector of FD phytoplasma, Scaphoideus titanus, has been identified in Montenegrin vineyards since 2008. During a survey in September 2011, in the northern part of Montenegro, 12 symptomatic alder trees showing symptoms of leaf discoloration, ranging from yellow to light green, were sampled. Six samples, each comprising several symptomatic leaves, were collected from A. glutinosa at streamside in woodlands near the town Kolasin and other six samples from A. incana close to the river Lim near the town of Bijelo Polje. Leaves of six young A. glutinosa seedlings were used as controls. Total DNA was extracted from fresh leaf midribs and petioles using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Nested PCR assay was conducted on 16S rRNA gene using phytoplasma generic primers P1/P7 and F2n/R2 followed by RFLP with MseI endonuclease (Fermentas, Vilnius, Lithuania) (3). Confirmation of identification and characterization of phytoplasma positive samples was performed by amplifying the non-ribosomal metionine aminopeptidase (map) gene using FD9f5/MAPr1 and FD9f6/MAPr2 primer set (1), specific for the members of the 16SrV group phytoplasmas. Amplification products were sequenced and deposited in GenBank (KC188998 through 9001). Comparison of the map gene sequences was performed by phylogenetic analysis along with 20 reference sequences of the 16SrV-group members (1), using the neighbor-joining method in MEGA5 software (4). 16S rRNA gene amplification revealed the presence of phytoplasmas in 11 out of 12 symptomatic samples, while Mse I restriction analysis and comparison with reference strains (AldYp and FDp from Serbia) enabled affiliation of detected phytoplasmas to the 16SrV-group. None of the controls were positive for any phytoplasma. Phylogenetic analysis of the Montenegrin AldYp map gene sequences revealed presence of four different strains clustering within the previously defined clusters of the 16SrV-group members (1). Three different strains associated with symptomatic A. glutinosa were identified and they clustered either within the FD1, FD2, or PGY-C cluster, while a single detected strain from A. incana proved to be identical with PGY-A isolate of AldY phytoplasma infecting grapevine in Germany (AM384892). To our knowledge, this is the first report of the association of 16SrV-group phytoplasmas with common and grey alder in Montenegro, as well as the first report of FD-related phytoplasmas in Montenegro. Since alder trees are considered as a possible natural reservoir of the FD phytoplasmas (1), the finding of alders naturally infected with strains related to the FDp (FD1 and FD2 clusters) indicate a possible threat of economic importance to the grape production in Montenegro, which should be addressed in further research. References: (1) G. Arnaud et al. Appl. Environ. Microbiol. 73:4001, 2007. (2) T. Cvrkovic et al. Plant Pathol. 57:773, 2008. (3) I-M. Lee et al. Int. J. Syst. Evol. Bacteriol. 48:1153, 1998. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

Endogenous ethanol production in patients with diabetes mellitus as a medicolegal problem.

Ethanol which is not ingested but is produced within the body through metabolic processes is known as "endogenous ethanol" (EnEth). The aim of this study was to determine whether there is a significant increase in the blood value of EnEth (BAC) in patients with diabetes mellitus and whether BAC correlates with increased glucose blood levels. In our study the BAC in the group of patients with diabetes mellitus (n=130) was significantly higher (mean value 2.65mg/L) than in the control group (mean value 0.40mg/L) when blood samples were analyzed by headspace gas chromatography method (HS-GC). The BAC levels obtained by semi-quantitative Widmark's method (n=60) were higher, and mean value in the patient group was 27.28mg/L. There was no correlation between the glucose blood levels and BAC, i.e. the auto-production of ethanol does not depend on blood glucose values. The mean value of ethanol in urine in patients with diabetes mellitus was 6.13mg/L measured by HS-GC method and 54.27mg/L when measured using Widmark's method. Even though the BAC in patients with diabetes mellitus was statistically significantly higher (p<0.01) in comparison to the control group, these amounts of EnEth do not increase greatly enough to affect legal proceedings, especially considering the fact that EnEth is measured in mg/mL. Our experimental results as well as data reported in the literature suggest that the cut-off level for the EnEth in blood should be set at BAC<0.1mg/mL for the semi-quantitative method, i.e. the values that appear at the second decimal place cannot be with certainty claimed to arise form the exogenous ethanol. For the HS-GS method cut-off level should be set at BAC<0.01mg/mL for the analytical parameters that were used in our experiments.