A novel thermostable YtnP lactonase from Stenotrophomonas maltophilia inhibits Pseudomonas aeruginosa virulence in vitro and in vivo.

Infections caused by multidrug-resistant pathogens are one of the biggest challenges facing the healthcare system today. Quorum quenching (QQ) enzymes have the potential to be used as innovative enzyme-based antivirulence therapeutics to combat infections caused by multidrug-resistant pathogens. The main objective of this research was to describe the novel YtnP lactonase derived from the clinical isolate Stenotrophomonas maltophilia and to investigate its antivirulence potential against multidrug-resistant Pseudomonas aeruginosa MMA83. YtnP lactonase, the QQ enzyme, belongs to the family of metallo-ß-lactamases. The recombinant enzyme has several advantageous biotechnological properties, such as high thermostability, activity in a wide pH range, and no cytotoxic effect. High-performance liquid chromatography analysis revealed the activity of recombinant YtnP lactonase toward a wide range of N-acyl-homoserine lactones (AHLs), quorum sensing signaling molecules, with a higher preference for long-chain AHLs. Recombinant YtnP lactonase was shown to inhibit P. aeruginosa MMA83 biofilm formation, induce biofilm decomposition, and reduce extracellular virulence factors production. Moreover, the lifespan of MMA83-infected Caenorhabditis elegans was prolonged with YtnP lactonase treatment. YtnP lactonase showed synergistic inhibitory activity in combination with gentamicin and acted additively with meropenem against MMA83. The described properties make YtnP lactonase a promising therapeutic candidate for the development of next-generation antivirulence agents.

Serotype distribution, antimicrobial susceptibility and molecular epidemiology of invasive Streptococcus pneumoniae in the nine-year period in Serbia.

Streptococcus pneumoniae is one of the leading bacterial pathogens that can cause severe invasive diseases. The aim of the study was to characterize invasive isolates of S. pneumoniae obtained during the nine-year period in Serbia before the introduction of the pneumococcal conjugate vaccines (PCVs) into routine vaccination programs by determining: serotype distribution, the prevalence and genetic basis of antimicrobial resistance, and genetic relatedness of the circulating pneumococcal clones. A total of 490 invasive S. pneumoniae isolates were included in this study. The serotype, antimicrobial susceptibility, and ST of the strains were determined by the Quellung reaction, disk- and gradient-diffusion methods, and multilocus sequence typing (MLST), respectively. The most common serotypes in this study were 3, 19F, 14, 6B, 6A, 19A, and 23F. The serotype coverages of PCV10 and PCV13 in children less than 2 years were 71.3 and 86.1%, respectively, while PPV23 coverage in adults was in the range of 85-96%, depending on the age group. Penicillin and ceftriaxone-non-susceptible isolates account for 47.6 and 16.5% of all isolates, respectively. Macrolide non-susceptibility was detected in 40.4% of isolates, while the rate of multidrug- and extensive-drug resistance was 20.0 and 16.9%, respectively. The MLST analysis of 158 pneumococci identified 60 different STs belonging to the 16 Clonal Complexes (CCs) (consisting of 42 STs) and 18 singletons. The most common CC/ST were ST1377, CC320, CC15, CC273, CC156, CC473, CC81, and CC180. Results obtained in this study indicate that the pre-vaccine pneumococcal population in Serbia is characterized by high penicillin and macrolides non-susceptibility, worrisome rates of MDR and XDR, as well as a high degree of genetic diversity. These findings provide a basis for further investigation of the changes in serotypes and genotypes that can be expected after the routine introduction of PCVs.

Comparative genomics of trimethoprim-sulfamethoxazole-resistant Achromobacter xylosoxidans clinical isolates from Serbia reveals shortened variant of class 1 integron integrase gene.

Trimethoprim-sulfamethoxazole (SXT) is the preferable treatment option of the infections caused by Achromobacter spp. Our study aimed to analyze the SXT resistance of 98 Achromobacter spp. isolates from pediatric patients, among which 33 isolates were SXT-resistant. The presence of intI1 was screened by PCR and genome sequence analyses. The intI1 gene was detected in 10 of SXT-resistant isolates that had shorter intI1 PCR fragments named intI1S. Structural changes in intI1S were confirmed by genome sequencing and analyses which revealed 86 amino acids deletion in IntI1S protein compared to canonical IntI1 protein. All IntI1S isolates were of non-CF origin. Pan-genome analysis of intI1S bearing A. xylosoxidans isolates comprised 9052 genes, with the core genome consisting of 5455 protein-coding genes. Results in this study indicate that IntI1S isolates were derived from clinical settings and that cystic fibrosis (CF) patients were potential reservoirs for healthcare-associated infections that occurred in non-CF patients.

B7 homologue 3 as a prognostic biomarker and potential therapeutic target in gastrointestinal tumors.

The most common digestive system (DS) cancers, including tumors of the gastrointestinal tract (GIT) such as colorectal cancer (CRC), gastric cancer (GC) and esophageal cancer (EC) as well as tumors of DS accessory organs such as pancreatic and liver cancer, are responsible for more than one-third of all cancer-related deaths worldwide, despite the progress that has been achieved in anticancer therapy. Due to these limitations in treatment strategies, oncological research has taken outstanding steps towards a better understanding of cancer cell biological complexity and heterogeneity. These studies led to new molecular target-driven therapeutic approaches. Different in vivo and in vitro studies have revealed significant expression of B7 homologue 3 (B7-H3) among the most common cancers of the GIT, including CRC, GC, and EC, whereas B7-H3 expression in normal healthy tissue of these organs was shown to be absent or minimal. This molecule is able to influence the biological behavior of GIT tumors through the various immunological and nonimmunological molecular mechanisms, and some of them are shown to be the result of B7-H3-related induction of signal transduction pathways, such as Janus kinase 2/signal transducer and activator of transcription 3, phosphatidylinositol 3-kinase/protein kinase B, extracellular signal-regulated kinase, and nuclear factor-κB. B7-H3 exerts an important role in progression, metastasis and resistance to anticancer therapy in these tumors. In addition, the results of many studies suggest that B7-H3 stimulates immune evasion in GIT tumors by suppressing antitumor immune response. Accordingly, it was observed that experimental depletion or inhibition of B7-H3 in gastrointestinal cancers improved antitumor immune response, impaired tumor progression, invasion, angiogenesis, and metastasis and decreased resistance to anticancer therapy. Finally, the high expression of B7-H3 in most common cancers of the GIT was shown to be associated with poor prognosis. In this review, we summarize the established data from different GIT cancer-related studies and suggest that the B7-H3 molecule could be a promising prognostic biomarker and therapeutic target for anticancer immunotherapy in these tumors.

Burkholderia cepacia YtnP and Y2-aiiA lactonases inhibit virulence of Pseudomonas aeruginosa via quorum quenching activity.

Burkholderia cepacia is well known as the causative agent of infections in humans where often shares niche with other pathogens, like Pseudomonas aeruginosa. Clinical isolate Burkholderia sp. BCC4135 was selected due to its strong quorum quenching (QQ) activity. Whole genome sequencing unveiled this isolate as B. cepacia with unique sequence type ST1485 and a myriad of genes belonging to resistome and virulome. Two QQ lactonases YtnP and Y2-aiiA originated from B. cepacia BCC4135 were cloned, expressed, and functionally characterized. They were active against a broad substrate spectrum of the N-acyl-homoserine lactones (AHLs). The YtnP lactonase was inactive, while Y2-aiiA was active against N-tetradecanoyl-dl-homoserine lactone (C14-HSL) which could imply the difference in their biological roles from the aspect of its quorum sensing (QS) autoregulation and interference with the QS systems of bacteria residing within the same niche. Both YtnP and Y2-aiiA were able to attenuate virulence potential of P. aeruginosa MMA83 clinical isolate declining its biofilm formation and virulence factors production. B. cepacia BCC4135 lactonases interfered with the las, rhl, and even pqs QS circuit of P. aeruginosa MMA83 transcription and the effect of combined enzymes was even more prominent. B. cepacia BCC4135 also employs the CepI/R QS system for governing its own virulence traits and possibly self-regulates the QQ/QS network through the different expression and activity of YtnP and/or Y2-aiiA. Our findings pointed out that BCC4135 lactonases could be exploited as an effective antivirulence drugs against P. aeruginosa and gave us a new insight into B. cepacia QQ/QS machinery.

Abiotrophia defectiva liver abscess in a teenage boy after a supposedly mild blunt abdominal trauma: a case report.

BACKGROUND: A pyogenic liver abscess (PLA) represents a pus-filled cavity within the liver parenchyma caused by the invasion and multiplication of bacteria. The most common offender isolated from the PLA in children is Staphylococcus aureus. Abiotrophia defectiva is a Gram-positive pleomorphic bacterium, commonly found in the oral cavity, intestinal, and genitourinary mucosa as part of the normal microbiota. It has been proven to be an etiological factor in various infections, but rarely in cases of PLA. The case presented here is, to the best of our knowledge, the first pediatric case of PLA caused by A. defectiva. CASE PRESENTATION: A 13-year-old Caucasian boy presented with a two-day history of abdominal pain, fever up to 40 °C, and polyuria. Contrast-enhanced computed tomography (CT) scan revealed a single, multiloculated liver lesion, suggestive of a liver abscess. The boy had sustained a bicycle handlebar injury to his upper abdomen 3 weeks before the symptoms appeared and had been completely asymptomatic until 2 days before admission. He was successfully treated with antibiotic therapy and open surgical drainage. A. defectiva was isolated from the abscess material. Histopathology report described the lesion as a chronic PLA. CONCLUSIONS: A. defectiva is a highly uncommon cause of liver abscess in children. In such cases, various predisposing factors should be considered, including antecedent blunt abdominal trauma.

Fluoroquinolone-resistant Achromobacter xylosoxidans clinical isolates from Serbia: high prevalence of the aac-(6')-Ib-cr gene among resistant isolates.

The aim of this study was to evaluate the contribution of plasmid-mediated genes and efflux to fluoroquinolone resistance in collection of Achromobacter spp. gathered during a 3-year period. Susceptibility to ciprofloxacin and levofloxacin was tested by disk diffusion and microdilution tests for a collection of 98 Achromobacter spp. clinical isolates. Identification of fluoroquinolone-resistant isolates was performed by sequencing and phylogenetic analyses of the nrdA gene. Genetic relatedness among resistant isolates was determined by pulsed-field gel electrophoresis (PFGE) analysis. The influence of an H+ conductor cyanide m-chlorophenyl hydrazone (CCCP) and a resistance-nodulation-division-type efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PAßN) on minimal inhibitory concentration (MIC) value was evaluated by broth microdilution. The presence of the plasmid-mediated qnrA, qnrB, qnrC, qnrS, and aac-(6')-Ib-cr genes was investigated by PCR and sequencing. Achromobacter spp. isolates that were resistant or intermediately resistant to fluoroquinolones in disk diffusion tests (44/98) were subjected to microdilution. As a result, 20/98 isolates were confirmed to be resistant to ciprofloxacin while 10/98 was resistant to levofloxacin. CCCP decreased twofold MIC value for ciprofloxacin in six isolates and more than 16 times in one isolate, while MIC value for levofloxacin was decreased in all isolates (twofold to more than eightfold). Fluoroquinolone-resistant isolates were identified as A. xylosoxidans with the nrdA gene sequencing. PFGE revealed that resistant isolates belonged to seven different genotypes. Ten isolates belonging to four genotypes were positive for the aac-(6')-Ib-cr gene. Although resistance to fluoroquinolones was not widespread among analyzed isolates, detected contribution of efflux pumps and the presence of the aac-(6')-Ib-cr gene present a platform for emergence of more resistant strains.

Candida bloodstream infections in Serbia: First multicentre report of a national prospective observational survey in intensive care units.

Candida bloodstream infections (BSI) are a significant cause of mortality in intensive care units (ICU), hereof the prospective 12-months (2014-2015) hospital- and laboratory-based survey was performed at the Serbian National Reference Medical Mycology Laboratory (NRMML). Candida identification was done by a matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and a susceptibility test, according to the Clinical and Laboratory Standards Institute methodology. Among nine centres (265 beds; 10 820 patient admissions), four neonatal/paediatric (NICU/PICUs) and five adult centres (ICUs) participated, representing 89 beds and 3446 patient admissions, 166 beds and 7347 patient admissions respectively. The NRMML received 43 isolates, 17 from NICU/PICUs and 26 from adult ICUs. C. albicans dominated highly in NICU/PICUs (~71%), whereas C. albicans and C. parapsilosis were equally distributed within adults (46%, each), both accounting for ~90% of received isolates. The resistance to itraconazole and flucytosine were 25% and 2.4% respectively. In addition, the 2 C. albicans were azole cross-resistant (4.6%). The overall incidence of CandidaBSI was ~3.97 cases/1000 patient admissions (4.93 in NICU/PICU and 3.53 in adult ICU). The 30-day mortality was ~37%, most associated with C. tropicalis and C. glabrataBSI. Data from this national survey may contribute to improving the Balkan and Mediterranean region epidemiology of CandidaBSI within ICUs.

Uncovering Differences in Virulence Markers Associated with Achromobacter Species of CF and Non-CF Origin.

Achromobacter spp. are recognized as emerging pathogens in hospitalized as well as in cystic fibrosis (CF) patients. From 2012 to 2015, we collected 69 clinical isolates (41 patient) of Achromobacter spp. from 13 patients with CF (CF isolates, n = 32) and 28 patients receiving care for other health conditions (non-CF isolates, n = 37). Molecular epidemiology and virulence potential of isolates were examined. Antimicrobial susceptibility, motility, ability to form biofilms and binding affinity to mucin, collagen, and fibronectin were tested to assess their virulence traits. The nrdA gene sequencing showed that A. xylosoxidans was the most prevalent species in both CF and non-CF patients. CF patients were also colonized with A. dolens/A. ruhlandii, A. insuavis, and A. spiritinus strains while non-CF group was somewhat less heterogenous, although A. insuavis, A. insolitus, and A. piechaudii strains were detected beside A. xylosoxidans. Three strains displayed clonal distribution, one among patients from the CF group and two among non-CF patients. No significant differences in susceptibility to antimicrobials were observed between CF and non-CF patients. About one third of the isolates were classified as strong biofilm producers, and the proportion of CF and non-CF isolates with the ability to form biofilm was almost identical. CF isolates were less motile compared to the non-CF group and no correlation was found between swimming phenotype and biofilm formation. On the other hand, CF isolates exhibited higher affinity to bind mucin, collagen, and fibronectin. In generall, CF isolates from our study exhibited in vitro properties that could be of importance for the colonization of CF patients.

Virulence traits associated with Burkholderia cenocepacia ST856 epidemic strain isolated from cystic fibrosis patients.

BACKGROUND: Burkholderia cenocepacia is considered one of the most problematic cystic fibrosis (CF) pathogens. Colonization prevalence in the Serbian CF population is high and virtually exclusively limited to a single highly transmissible clone of B. cenocepacia ST856 which is positive for both the B. cepacia epidemic strain marker (BCESM) and cable pilin, and is closely related to the epidemic strain CZ1 (ST32). METHODS: Biofilm formation for 182 isolates, and adhesion to components of the host extracellular matrix, proteolytic activity, mucoidy and motility of selected ST856 representatives, as well as B. cenocepacia ST858 and ST859, and B. stabilis ST857, novel STs isolated from Serbian CF patients, were investigated in this study. The presence of the cepI, cepR, fliG, llpE, wbiI, and bcscV genes was analyzed. RESULTS: Biofilm-formation ability of analyzed strains was poor under standard laboratory conditions, but changed in stress conditions (cold stress) and conditions that mimic CF milieu (increased CO2). All strains expressed ability to bind to collagen and fibronectin albeit with different intensity. Representatives of ST856 exhibited gelatinase activity. ST858, ST859 and 9/11 of ST856 genotypes were positive for swimming and twitching motility whereas ST857 was non-motile. Mucoidy was demonstrated in all ST856 genotypes, ST857 was semi-mucoid, and ST858 and ST859 were non-mucoid. Molecular analysis for major virulence factors revealed that ST856 and ST857 carried the six analyzed genes, while ST858 and ST859 were negative for the llpE gene. CONCLUSION: Variations in virulence phenotypes in different genotypes of epidemic B. cenocepacia ST856 clone, in vitro, could be a consequence of diversification driven by pathoadaptation. Diversity of epidemic clone genotypes virulence, could be challenging for accurate diagnosis and treatment, as well as for infection control.

Molecular Epidemiology of Colistin-Resistant, Carbapenemase-Producing Klebsiella pneumoniae in Serbia from 2013 to 2016.

Twenty-seven colistin-resistant, carbapenemase-producing Klebsiella pneumoniae isolates were identified from hospitals in Serbia. All isolates were blaCTX-M-15 positive; ST101, ST888, ST437, ST336, and ST307 were blaOXA-48 positive; and ST340 was blaNDM-1 positive. ST307 had an insertion, and ST336 had a premature stop codon in the mgrB gene. Amino acid substitutions were detected in PmrAB of isolates ST101, ST888, ST336, and ST307. The mcr-1 and mcr-2 were not detected. An increase in phoP, phoQ, and pmrK gene transcription was detected for all sequence types.

Temperature, pH and Trimethoprim-Sulfamethoxazole Are Potent Inhibitors of Biofilm Formation by Stenotrophomonas maltophilia Clinical Isolates.

Stenotrophomonas maltophilia, an opportunistic pathogen usually connected with healthcare-associated infections, is an environmental bacterium. Intrinsic resistance to multiple antibiotics, with different virulence determinants in the last decade classified this bacterium in the group of global multiple drug resistant (MDR) organism. S. maltophilia clinical isolates, were collected from tertiary care pediatric hospital in Belgrade, Serbia to investigate influence of different factors on biofilm formation, kinetics of biofilm formation for strong biofilm producers and effect of trimethoprim-sulfamethoxazole (TMP/SMX) on formed biofilm. Most of the isolates (89.8%) were able to form a biofilm. Analysis of biofilm formation in different growth conditions showed that changing of temeperature and pH had the stronggest effect on biofilm formation almost equally in group of cystic fibrosis (CF) and non-CF strains. TMP/SMX in concentration of 50 µg/ml reduced completely 24 h old biofilms while concentration of 25 µg/ml effects formed biofilms in a strain dependent manner. Among strains able to form strong biofilm CF isolates formed biofilm slower than non-CF isolates, while shaking conditions did not affect biofilm formation. Swimming motility was detected in both CF and non-CF isolates, however more motile strain formed stronger biofilms. This study suggests that temperature, pH and TMP/SMX had the strongest influence on biofilm formation in analyzed collection of S. maltophilia. A positive correlation between motility and strength of formed biofilm was demonstrated.

Genotypic and Phenotypic Characterization of Stenotrophomonas maltophilia Strains from a Pediatric Tertiary Care Hospital in Serbia.

BACKGROUND: Stenotrophomonas maltophilia is an environmental bacterium and an opportunistic pathogen usually associated with healthcare-associated infections, which has recently been recognized as a globally multi-drug resistant organism. The aim of this study was genotyping and physiological characterization of Stenotrophomonas maltophilia isolated in a large, tertiary care pediatric hospital in Belgrade, Serbia, hosting the national reference cystic fibrosis (CF) center for pediatric and adult patients. METHODS: We characterized 42 strains of cystic fibrosis (CF) and 46 strains of non-cystic fibrosis (non-CF) origin isolated from 2013 to 2015 in order to investigate their genetic relatedness and phenotypic traits. Genotyping was performed using sequencing of 16S rRNA gene, Pulse Field Gel Electrophoresis (PFGE) and Multi locus sequencing typing (MLST) analysis. Sensitivity to five relevant antimicrobial agents was determined, namely trimethoprim/sulfamethoxazole (TMP/SMX), chloramphenicol, ciprofloxacin, levofloxacin and tetracycline. Surface characteristics, motility, biofilm formation and adhesion to mucin were tested in all strains. Statistical approach was used to determine correlations between obtained results. RESULTS: Most of the isolates were not genetically related. Six new sequence types were determined. Strains were uniformly sensitive to all tested antimicrobial agents. The majority of isolates (89.8%) were able to form biofilm with almost equal representation in both CF and non-CF strains. Swimming motility was observed in all strains, while none of them exhibited swarming motility. Among strains able to adhere to mucin, no differences between CF and non-CF isolates were observed. CONCLUSIONS: High genetic diversity among isolates implies the absence of clonal spread within the hospital. Positive correlation between motility, biofilm formation and adhesion to mucin was demonstrated. Biofilm formation and motility were more pronounced among non-CF than CF isolates.

Novel E. coli ST5123 Containing blaNDM-1 Carried by IncF Plasmid Isolated from a Pediatric Patient in Serbia.

New Delhi metallo-ß-lactamase (NDM) is a serious challenge to the treatment of infections and public health. Serbia has been designated as an endemic region for isolates carrying the blaNDM-1 gene, as well as one of several commonly proposed countries of origin. This is the first report of NDM-1-positive Escherichia coli from Serbia. A carbapenem-resistant clinical isolate of E. coli strain IMD989, isolated from the blood culture of a pediatric patient with leukemia, was subjected to antimicrobial susceptibility tests, molecular typing, and conjugation experiments. The strain exhibited resistance to meropenem and was classified as a novel sequence type, ST5123, belonging to E. coli phylogenetic group A. ST5123 showed similarity to veterinary isolates ST93 and ST3977. The blaNDM-1 gene was detected by polymerase chain reaction (PCR) and sequencing. Cloning and sequencing of genomic clones confirmed that strain IMD989 produces an NDM-1 variant. Conjugation experiments, pulsed-field gel electrophoresis, and Southern blot hybridization revealed that blaNDM-1 was located in IMD989 on a transmissible 80 kb plasmid, designated as pIMD989. PCR analysis confirmed that pIMD989 belongs to the IncF plasmid family. Propagation of IMD989 and selected transconjugants carrying pIMD989 over 14 days in solid media with and without antibiotic selection showed that pIMD989 is a stable plasmid.

Carbapenem-resistant Acinetobacter baumannii from Serbia: revision of CarO classification.

Carbapenem-resistant A. baumannii present a significant therapeutic challenge for the treatment of nosocomial infections in many European countries. Although it is known that the gradient of A. baumannii prevalence increases from northern to southern Europe, this study provides the first data from Serbia. Twenty-eight carbapenem-resistant A. baumannii clinical isolates were collected at a Serbian pediatric hospital during a 2-year period. The majority of isolates (67.68%) belonged to the sequence type Group 1, European clonal complex II. All isolates harbored intrinsic OXA-51 and AmpC cephalosporinase. OXA-23 was detected in 16 isolates (57.14%), OXA-24 in 23 isolates (82.14%) and OXA-58 in 11 isolates (39.29%). Six of the isolates (21.43%) harbored all of the analyzed oxacillinases, except OXA-143 and OXA-235 that were not detected in this study. Production of oxacillinases was detected in different pulsotypes indicating the presence of horizontal gene transfer. NDM-1, VIM and IMP were not detected in analyzed clinical A. baumannii isolates. ISAba1 insertion sequence was present upstream of OXA-51 in one isolate, upstream of AmpC in 13 isolates and upstream of OXA-23 in 10 isolates. In silico analysis of carO sequences from analyzed A. baumannii isolates revealed the existence of two out of six highly polymorphic CarO variants. The phylogenetic analysis of CarO protein among Acinetobacter species revised the previous classification CarO variants into three groups based on strong bootstraps scores in the tree analysis. Group I comprises four variants (I-IV) while Groups II and III contain only one variant each. One half of the Serbian clinical isolates belong to Group I variant I, while the other half belongs to Group I variant III.

Chronic necrotizing pulmonary aspergillosis in an immunocompetent, obese 10-year-old boy.

Chronic necrotizing pulmonary aspergillosis is a rare form of pulmonary aspergillosis. It is usually seen in middle-aged or elderly patients with underlying chronic lung disease or mild immunodeficiency, and has been only rarely encountered in children. Clinical presentation is variable and usually involves constitutional symptoms of several months' duration as well as respiratory symptoms. We present a previously well, immunocompetent, obese 10-year-old boy with cough and mild hemoptysis lasting for a couple of days and a round pulmonary infiltrate on chest radiograph. Further diagnostic investigations revealed the histopathological features of chronic necrotizing pulmonary aspergillosis in excised lung tissue, and Aspergillus fumigatus was isolated in lung tissue culture. This is one of the youngest described patients with this semi-invasive form of aspergillosis.

Atypical strain of Toxoplasma gondii causing fatal reactivation after hematopoietic stem cell transplantion in a patient with an underlying immunological deficiency.

In immunocompromized patients, including hematopoietic stem cell transplant (HSCT) recipients, life-threatening toxoplasmosis may result from reactivation of previous infection. We report a case of severe disseminated toxoplasmosis that developed early after allogeneic HSCT for T-cell lymphoblastic leukemia/lymphoma in a 15-year-old Toxoplasma gondii-seropositive boy with Nijmegen breakage syndrome, a rare genetic DNA repair disorder associated with immunodeficiency. The donor was the patient's HLA-identical brother. Prophylaxis with cotrimoxazole was discontinued a day before the HSCT procedure. Signs of lung infection appeared as early as day 14 post-HSCT. The presence of tachyzoite-like structures on Giemsa-stained bronchoalveolar lavage (BAL) fluid smears suggested toxoplasmosis. Real-time PCR targeted at the T. gondii AF146527 gene revealed extremely high parasite burdens in both blood and BAL fluid. Although immediate introduction of specific treatment resulted in a marked reduction of the parasite load and transient clinical improvement, the patient deteriorated and died of multiple organ failure on day 39 post-HSCT. Direct genotyping of T. gondii DNA from blood and BAL fluid with the PCR-restriction fragment length polymorphism method revealed type II alleles with SAG1, SAG2, and GRA6 markers but alleles of both type I and type II with GRA7. Additional analysis with 15 microsatellite markers showed that the T. gondii DNA was atypical and genetically divergent from that of the clonal type I, II, and III strains. This is the first report of increased clinical severity of toxoplasmosis associated with an atypical strain in the setting of immunosuppression, which emphasizes the need to diagnose and monitor toxoplasmosis by quantitative molecular methods in cases of reactivation risk.

Clinical characteristics of respiratory syncytial virus infection in neonates and young infants.

INTRODUCTION/AIM: Infection with respiratory syncytial virus (RSV) occurs during the first year of life in 50% of children and 20%-40% of them have signs of lower respiratory tract infection (bronchiolitis or pneumonia). There is an increased risk for complicated course and death from RSV infection in premature infants, especially those with bronchopulmonary dysplasia (BPD) or congenital heart disease. The aim of our study was to analyze clinical characteristics of laboratory confirmed RSV infection in order to evaluate the need for preventive measures in neonates and young infants. METHODS: The prospective study included children under age of 12 months admitted to our hospital in the period November 2008-March 2009 who were positive for RSV by enzyme immunoassay membrane test. The course of disease was assessed by clinical score and radiographic findings. RESULTS: Infection with RSV was confirmed in 91 patients: 21 (23.0%) were under the age of 30 days, 37 (40.7%) were between 31-60 days, and 33 patients (36.3%) were older than 60 days (p > 0.05). The highest hospitalization rate was in January--33 patients (36.3%; p < 0.01). Disease severity score in these age groups (AG) were: 8.4 +/- 0.4 (AG 0-30 days); 9.0 +/- 0.3 (AG 31-60 days) and 8.3 +/- 0.3 (AG > 60 days), without statistically significant difference among the groups (p > 0.05). Clinical scores in patients with and without risk factors were 10.5 +/- 0.5 and 8.3 +/- 0.2, respectively (p < 0.01). Pathological radiographic findings were observed in 72 (79.1%) and complications (apnea, significant atelectasis, encephalopathy) occured in 15 (16.5%) patients. The average length of hospital stay in complicated and uncomplicated course of the disease was 9 days and 6 days, respectively (p < 0.01). Therapy in 85 (93.4%) patients included bronchodilators, while systemic glucocorticoids and oxygen therapy were used in 51 (56.0%) and 44 (48.4%) patients, respectively. Death occured in 2 (2.2%) patients, both from a high risk group (the patient with BPD and the other one with congenital heart disease and Down syndrome). CONCLUSION: Infection with RSV in our settings showed marked seasonal characteristics with highest hospitalization rate in January. Although the course and outcome of the disease were favorable in the majority of our patients, the need for hospitalization and administration of therapy with possible side effects warrants that general measures for prevention of respiratory infections are followed especially in the first year of life. Severe disease and death are more probable in neonates and infants with risk factors. In these children passive immunisation with specific monoclonal antibody (e.g. palivizumab) during RSV season should be considered.

Neonatal cellulitis and sepsis caused by group A streptococcus.

We report a case of late onset neonatal invasive group A streptococcal disease characterized with rapidly progressing cellulitis and development of sepsis. The infection was acquired from benign and mild skin infection of the child's mother. The causative agent was group A streptococcus, belonging to the emm type 53.2, which usually causes mild skin disease.