First detection of human metapneumovirus in children with respiratory infections in Romania

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BACTERIAL PRODUCT CANTASTIM DERIVED FROM PSEUDOMONAS AERUGINOSA INDUCES MIGRATION AND MATURA-TION OF DENDRITIC CELLSIuliana Caraº, Cãtãlin Þucureanu, Lucian Lerescu and Aurora Sãlãgeanu

SYSTEMIC INFLAMMATORY MARKERS IN PATIENTS WITH AORTIC SCLEROSISMihaela Rugina, Iuliana Caras, Ruxandra Jurcut, Ciprian Jurcut, Francisca Serbanescu, Aurora Salageanu and Eduard Apetrei

ADJUVANT PROPERTIES OF BACTERIAL PRODUCT CANTASTIM Iuliana Francisca Anghelache, Iuliana Caras and Aurora Salageanu

CHARACTERIZATION OF GUANYLATE KINASE FROM GRAM POSITIVE AND GRAM NEGATIVE MICROORGANISMS;PRELIMINARY RESULTSAna-Maria Ruxandra Eftimie, Florina Toma, Adriana-Zoe Costache and Nadia Bucurenci

CONTROL OF BLOOD-TRANSMITTED INFECTIONS IN DENTISTRYEugenia Aurora Negut, Monica Balteanu, G. Ionescu, A. Bancescu, A. Iliescu and N. Skaug

FIRST DETECTION OF HUMAN METAPNEUMOVIRUS IN CHILDREN WITH RESPIRATORY INFECTIONS IN ROMANIACristina Tecu, D. Orasanu, Aurora Sima, Maria Elena Mihai, V. Alexandrescu, D. Matei and Narcisa Samoila

VIRULENCE CHARACTERISTICS OF ESCHERICHIA COLI ISOLATES FROM CHILDREN WITH URINARY TRACT INFECTIONSCaliopsia Florea, Codruta-Romanita Usein, Maria Condei and Maria Damian

THE MAINTAINING OF THE ACTIVE LABORATORY-BASED SURVEILLANCE OF THE ACUTE FLACCID PARALYSIS (AFP)CASES IN ROMANIA IN THE FRAMEWORK OF THE STRATEGIC PLAN OF THE GLOBAL POLIO ERADICATION INITIATIVEAnda Baicus, Ana Persu, Mariana Combiescu and A. Aubert-Combiescu

CONTENTS

IMMUNOLOGY

MICROBIOLOGY

VOLUME 66 NOS. 1-2 JANUARY - JUNE 2007

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Aims and ScopeRomanian Archives of Microbiology and Immunoloy, an internation-al journal dedicated to original research work, publishes papers focus-ing on various aspects of microbiology and immunology. RomanianArchives of Microbiology and Immunology is indexed in MEDLINE.The frequency of the Journal is currently four issues per year.

Categories of manuscriptsFull-length articles are full-length descriptions of original research(up to 10 printed pages)Reviews are comprehensive appraisals of research in a field of currentinterest. All reviews are subject to the normal review process (up to15 printed pages)Rapid Communications are brief, definitive reports of highly signi-ficant and timely findings in the field (up to 5 printed pages)

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INSTRUCTIONS TO AUTHORS

INTRODUCTION

Dendritic cells are the most potent antigen-presen-ting cells (APCs) that initiate and amplify immuneresponses. DCs progenitors in the bone marrow giverise to circulating precursors that home to tissues,where they reside as immature cells which act asimmunological sensors to alert for potentially dange-rous microbes. Immature DCs express members of theToll-like receptor (TLR) family, which bind commonchemical moieties associated with microbial orga-nisms. TLR ligands include bacterial lipopolysaccha-ride, lipopeptides, unmethylated CpG DNA motifs, dsRNA and flagellin. The recognition and internalizationof microbial structures induces the maturation of DCs,a process that is accompanied by their migration fromthe peripheral tissues into the T cell areas of draininglymph nodes. In the course of maturation, DCs aresubject to profound changes. The endocytic capacity isdownregulated, while there is a marked up-regulationof MHC class II expression. In addition, mature DCsexpress enhanced surface levels of co-stimulatory andadhesion molecules such as CD80, CD86 and CD40.As a result of these multiple modifications, mature DCs

ABSTRACTDendritic cells (DCs) play a pivotal role in linking innate and adaptive immunity. Migration to the lymphnodes and maturation of DCs are crucial steps in the initiation of specific immune responses. The bacte-rial product CANTASTIM (CS) is a purified extract of Pseudomonas aeruginosa that induces non-specificprotection against bacterial infection, enhances macrophage effector functions and modulates cytokinesproduction. In this study, we used a mouse skin explant culture model and human monocyte-derived DCsto study the effect of CS on the migration and maturation of DCs, respectively. We noticed a significantincrease in the number of DCs which migrated from the skin explants when CS was added to the culturemedium. Also, CS was able to induce the expression of maturation-associated marker CD83 on humanmonocyte-derived DCs. DC-based tumor vaccines represent a promising approach for cancer immunothe-rapy and the migration rate and maturation state of DCs are important parameters for their clinical effec-tiveness. CS may be an attractive candidate to be tested for the production of DC-based vaccine.

acquire the distinct ability to trigger a primary T cellresponse. Moreover, DCs play a critical role in thepolarization of T cell responses. Their activation bymicrobial antigens induces the production of cytokinessuch as IL-12, IL-18 or IL-10, which may promote thedevelopment of either type 1 T helper (Th1) cells ortype 2 T helper (Th2) cells [1-4].

CS is a second generation bacterial immunomodu-lator, prepared by ethanol extraction from a strain ofPseudomonas aeruginosa and further purified by ace-tone precipitation. The product is a mixture of bacteri-al components, comprised mainly of lipids (>80%),lipoproteins, lipopeptides, muramylpeptides and neu-tral sugars. Experimental studies in vivo and in vitrodemonstrated that CS has non-specific protective effect(prophylactic and therapeutic) against bacterial infec-tions, protective effect against endotoxin shock, acti-vates monocytes/macrophages, T lymphocytes and NKcells [5, 6].

The aim of this study was to assess the effect of CSon migration of DCs from murine skin explant and onmaturation of DCs derived from human peripheralblood monocytes.

BACTERIAL PRODUCT CANTASTIM DERIVED FROM PSEUDOMONAS AERUGINOSA INDUCES MIGRATION

AND MATURATION OF DENDRITIC CELLS

Iuliana Caraº*, Cãtãlin Þucureanu, Lucian Lerescu and Aurora Sãlãgeanu

Infection and Immunity Laboratory, „Cantacuzino“ National Institute of Research and Development for Microbiology and Immunology, Bucharest, Romania

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*Corresponding author: E-mail: [email protected]

Key words: dendritic cell, maturation, migration, CANTASTIM

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CARAª et al.

MATERIALS AND METHODS

In all cases when biological samples were neededfrom humans the procedures were approved by theCantacuzino Institute ethical committee and all volun-teers gave their informed consent.

MiceBALB/c mice bred in our own facilities were used

at the age of 6-8 weeks.

Skin organ cultureEars from mice were rinsed with 70% ethanol and

air dried for 10 min. Ear skin was split in dorsal andventral halves, and the dorsal halves were cultured ina 24-well tissue culture plate in 1.5 ml of RPMI 1640(Sigma) supplemented with 10% fetal calf serum (FCS,InVitrogen), 1 mM L-glutamine, 100 U/mL penicillin,100 µg/mL streptomycin and 10-7M ß-mercaptoetha-nol in the presence or absence of CS (20 µg/mL,Cantacuzino Institute). The tissue was transferred ontoculture wells with fresh medium ± CS every day. Thestandard culture was 3 days. The cells in the wells fromwhich the tissue had been removed and transferred onday 2 of culture were cultured until day 3. On day 3,the ear halves were removed and cells from day 2 and3 were pooled, centrifuged, and counted. DCs wereidentified morphologically as large veiled cells.

At least six explants (i.e. six wells) were pooled foreach experimental condition.

Generation of human monocyte-derived DCs(moDCs)

Peripheral blood mononuclear cells (PBMC) wereisolated from healthy volunteers by standard densitygradient centrifugation (30 min, 2000 rpm, 21oC,Histopaque®-1077, Sigma) and washed three timeswith complete culture medium (RPMI-1640 containing10% FCS, 1mM L-glutamine, 100U/mL penicillin and100 µg/mL streptomycin), each followed by centrifu-gation 10 min at 1500, 1200 and 1000 rpm, respec-tively to reduce platelet contamination. PBMC wereresuspended in complete culture medium, counted,plated at 10 x 106 / well (COSTAR® 6-well flat bottomplates, Corning Inc.) and incubated 2 h (37oC, 5%CO2) to allow monocyte adherence. After 2 h incuba-tion the nonadherent lymphocytes were removed andadherent monocytes were cultured in complete cul-ture medium supplemented with GM-CSF and IL-4[(60 ng/mL and 30 ng/mL, respectively), R&D Systems].Cultures were fed every other day by removing 0.8 mlof the medium and adding back 1 ml fresh mediumwith cytokines. The resulting cells exhibited DC mor-phology and clustering. Day 6 immature DCs wereused for further tests.

Induction of DCs maturationMoDCs were pooled on day 6, the concentration

adjusted to 5 x 105 cells/mL and the cells reculturedwith maturation factor in GM-CSF and IL-4 supple-mented medium for an additional 2 days. The matura-tion factor used as positive control was LPS 10 µg/mL(E. coli 055:B5 strain, Sigma-Aldrich Inc).

To test the ability of CS to activate immature DCs,different concentrations of CS (1, 10, 20 µg/mL) wereadded to the GM-CSF and IL-4 supplemented medium.After 48 h stimulation, phenotypic consequences ofDCs treatment with CS were evaluated.

Flow cytometry analysisFor the phenotypic analysis, fluoroscein isothio-

cyanate (FITC) or phycoerythrin (PE) conjugated mon-oclonal antibodies (mAbs) against following moleculeswere used: CD14, CD1a, CD83 (BD Pharmingen).Cells were stained for 30 min at 4oC and washed twicein PBS+1% BSA. Stained DCs were analyzed on FACSCalibur cytometer (Becton Dickinson) using CellQuest software. 5,000 gated cells were analyzed foreach parameter.

Statistic analysisStatistical analyses were performed using a paired

Student's t-test. P-values of less than 0.05 were con-sidered significant.

RESULTS

CS influences the migration of DCs from skinexplants into the culture medium

DCs emigrate spontaneously from murine wholeskin explants into the culture medium over a period of1-3 days. In the present study we used the skin organculture system described by Ortner et al. with slightmodifications [7]. Most of the cells harvested from theexplant cultures had the typical morphologic DCappearance of large veiled cells (Figure 1).

To assess the influence of CS on DCs migration, weadded CS (20 µg/mL) in the culture medium. The opti-mal stimulation dose was established in preliminaryexperiments. A concentration of CS lower than 20 µg/mLhad no significant influence on the migration of DCs(data not shown). When 20 µg/mL CS were added tothe cultures, a significant increase in the number ofDCs retrieved from the culture medium as comparedto unstimulated wells was noticed (Table 1).

Generation of immature moDCsDCs were generated from human PBMC in medi-

um containing cytokines GM-CSF and IL-4 for 6 days.Immature day 6 DCs displayed a typical morphology

CANTASTIM induces migration and maturation of dendritic cells

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inflammatory mediators such as LPS, TNF-α and IL-1ß.TNF-α downregulates E-cadherin on epidermalLangerhans cells, thereby loosening these cells withinthe epidermis and rendering them responsive tochemotactic stimuli such as macrophage inflammatoryprotein-3ß (CCL19) and secondary lymphoid tissuechemokine/CCL21 [10, 11].

In this study, we investigated the effect of bacterialimmunomodulator CS on the migration of DCs in skinexplant cultures. When CS was added to the culturemedium there was a significant increase in the numberof DCs that emigrated in the culture medium. Our groupstudies showed that, in vitro, CS activated PBMC tosecrete a variety of cytokines such as TNF-α, IL-1ß, IL-6,IL-10 and GM-CSF [5]. We may hypothesize that CScould promote the migration of DCs either directly orindirectly (e.g. by inducing cytokines secretion by cellsfrom the skin such as fibroblasts).

The maturation of DCs can be triggered by a vari-ety of factors, including microbial molecules (such asLPS from outer membranes of Gram-negative bacteria,lipoteichoic acids from gram-positive bacteria, CpG-containing DNA, bacterial flagellin etc.) componentsof necrotic cells or tissue matrices and products of acti-

(Figure 2A). The phenotypic analysis revealed an increa-se in the number of cells expressing CD1a marker (about47% of the cells) as well as a down-regulation of themonocyte marker CD14 (Figure 2B).

CS induces maturation of human moDCsTo investigate the effects of CS on the maturation of

moDCs, day 6 moDCs were treated with different con-centrations of CS (1, 10, 20 µg/mL). These doses did notcause a severe reduction of DCs viability (data notshown). As a positive control, moDCs were stimulatedwith LPS (10 µg/mL) known for its maturation-inducingcapacity [8]. Two days later, the phenotype of themoDCs was assessed by measuring the expression ofthe maturation-associated marker CD83. When moDCswere stimulated with LPS, 49.8% of the cells expre-ssed CD83 on their surface. Phenotypic analyses re-vealed that CS at 1 and 10 µg/mL had no influence onthe maturation of moDCs (data not shown). Stimu-lation of DCs with 20 µg/mL induced an increase ofCD83 positive cells from 8.8% (unstimulated cells) to18.9 % (Figure 3).

DISCUSSION

DCs reside in all body tissues, including the epithe-lia of the skin, lung, intestine, and urinary tract, themost likely entry sites of pathogens. DCs pick up anti-gens through fluid-phase and receptor mediated endo-cytosis. In addition, DCs respond to contact with pa-thogens through distinct receptors. Signaling throughthese receptors induces DCs maturation. ActivatedDCs become migratory and home to adjacent lymphnodes, where they activate T cells specific for bacterialor viral antigens. In addition, DCs support Th cell-dependent B-cell activation and thus the induction ofhumoral responses [9]. Maturation and migration ofDCs are tightly linked processes. Migration can appa-rently not occur without concomitant maturation.Dendritic cell migration is initiated and regulated by

Figure 1. DCs migrated from skin explants.Morphology of cells migrated from skin explants in cul-ture medium after 3 days culture; (magnification x 400).

Table 1 - Absolute numbers of migrated DCs from skin explants

a Skin explants were cultured for 3 days in medium ± CS, after which migrated DCs from day 2 and 3 from at least6 explants were pooled and quantified.

bThe values are the mean of cells emigrated per one explant from three experiments performed in similar conditions;*significant differences in paired Student's t-test: medium vs CS, p < 0.05.

Figure 2. Monocytes cultured with GM-CSF / IL-4 differentiate into immature DCs.

A. Morphology of day 6 immature DCs (magnification x 400). B. Phenotype of immature DCs: two-color cytograms ofday 6 cells labeled with FITC-CD14 and PE-CD1a mAbs;the horizontal and vertical lines indicate the level of auto-fluorescence in the FL-1 and FL-2 channels. Immature DCswere gated like large granular cells on the forward and sidescatter dot plot. One representative experiment of threeindependent experiments with a similar outcome is shown.

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CARAª et al.

vated leukocytes [12]. In humans, the DCs maturationprocess correlates with upregulation of CD83, a glyco-protein in the Ig superfamily bearing significanthomologies with the B7 gene family. Expression ofmembrane CD83 is restricted to the myeloid compart-ment and most prominently on DCs and thereforewidely used as a marker for this population [13].

Immature DCs, similar to those found in peripheraltissues, can be generated by culturing human mono-cytes with GM-CSF and IL-4 and have been used toidentify the activation signals that induce DCs matura-tion [14, 15].

In order to examine the maturation effect of CS onDCs, moDCs which had been cultured for 6 days,

Figure 3. CD83 expression on moDCs.Day 6 moDCs were left unstimulated for 48h or were treatedwith CS at 20 µg/mL or with LPS at 10 µg/mL. CD83 expres-sion was assessed by flow cytometry. Open histogramsindicate control labeling with an irrelevant mAb, whilesolid histograms indicate staining with anti-CD83 mAb.The values indicated on the histograms represent the per-centage of positive cells in the gated population. One re-presentative experiment of three independent experimentswith a similar outcome is shown.

were incubated for 48 h in the presence of CS and theexpression of maturation marker CD83 was measuredon the surface of moDCs. The results indicated that CSinduced a marked increase of the percentage of CD83positive moDCs. No significant change in cell viabilitywas observed, indicating that the effect was the resultof DCs maturation rather than a differential survival.

Dendritic cells represent an attractive vector foranti-cancer immunotherapy. In clinical protocols,tumor antigen-charged autologous DCs had beenadministered intracutaneously (s.c. or intradermally).They are expected to migrate to the draining lymphnodes and to induce immunity there but it has beenshown that a vast majority of DCs remain at the injec-tion site in the skin. Therefore, improvement of themigration rate of these cells could directly contributeto the efficiency of vaccination. Maturation state ofDCs is also a crucial parameter for clinical effective-ness of DC-based immunotherapy and DCs maturationstep should be optimized in future trials in order toimprove clinical responses [11, 16]. Our results clear-ly demonstrate that CS induces the migration and mat-uration of DCs. As this bioproduct has been on theRomanian market for many years, it may be an attrac-tive candidate to be tested for production of DC-basedvaccine.

AknowledgementsThe present study was supported by a grant from

BIOTECH Program for Research (CEEX 6/2005). The skillful technical assistance of Camelia Tabarta

is gratefully acknowledged.

REFERENCES

1. Banchereau J, Briere F, Caux C, Davoust J,Lebecque S, Liu YJ, Pulendran B, and Palucka K.Immunobiology of dendritic cells. Annu RevImmunol. 2000. 18:767-811

2. Moll H. Dendritic cells as a tool to combat infec-tious diseases. Immunol Lett. 2003. 85:153-157

3. Moll H. Dendritic cells and host resistance to infec-tion. Cell Microbiol. 2003. 5(8): 493-500

4. López R M and Moser M. Dendritic cell subsets andthe regulation of Th1/Th2 responses SeminarsImmunol 2001. 13: 275-282

5. Lerescu L, Tucureanu C, Caras I, and Salageanu A.Cytokine Profiling by Multiplex Immunoassay as anEffective Approach to Assess ImmunomodulatoryActivity of Bacterial Product CANTASTIM. RomArch Microbiol Immunol. 2006. 1-2:53-58

6. Caras I, Serbanescu IF., Grigorescu A, and Sala-geanu A. Experimental studies on bacterial product

CANTASTIM derived from Pseudomonas aerugi-nosa. VII. Activation of immune cells of healthycontrols and cancer patients. Roum Arch MicrobiolImmunol 2005. 64(1-4): 5-10.

7. Ortner U, Inaba K, Koch F, Heine M, Miwa M,Schuler G, and Romani N. An improved isolationmethod for murine migratory cutaneous dendriticcells. J Immunol Methods 1996.193(1): 71-79.

8. Palucka KA, Taquet N, Sanchez-Chapuis F, andGluckman JC. Dendritic Cells as the TerminalStage of Monocyte Differentiation. J Immunol1998. 160: 4587-4595.

9. Schmidhammer S, Ramoner R, Holtl L, Bartsch G,Thurnher M, and Zelle-Rieser C. An Escherichiacoli-based oral vaccine against urinary tract infec-tions potently activates human dendritic cells.Urology 2002. 60(3): 521-526.

10. Stoitzner P, Zanella M, Ortner U, Lukas M, Tag-werker A, Janke K, Lutz MB, Schuler G,Echtenacher B, Ryffel B, Koch F, and Romani N.Migration of Langerhans cells and dermal dendriticcells in skin organ cultures: augmentation by TNF-a and IL-1b. J Leukoc Biol 1999. 66: 462-470.

11. Ratzinger G, Stoitzner P, Ebner S, Lutz MB, Lay-ton GT, Rainer C, Senior RM, Shipley JM, FritschP, Schuler G, and Romani N. Matrix Metalloprotei-nases 9 and 2 Are Necessary for the Migration ofLangerhans Cells and Dermal Dendritic Cells fromHuman and Murine Skin. J Immunol 2002. 168:4361- 4371.

12. Skoberne M, Beignon AS, and Bhardwaj N. Dangersignals: a time and space continuum. Trends MolMed 2004. 10 (6): 251-257.

13. Li J, Mbow ML, Sun L, Li L, Yang G, Griswold DE,Schantz A, Shealy DJ, Goletz TJ, Wan J, Peritt D.Induction of dendritic cell maturation by IL-18. CellImmunol 2004. 227(2):103-108.

14. Sallusto F, and Lanzavecchia A. Efficient presenta-tion of soluble antigen by cultured human dendrit-ic cells is maintained by granulocyte/macrophagecolony-stimulating factor plus interleukin 4 anddownregulated by tumor necrosis factor. J Exp Med1994. 179:1109-1118.

15. Sallusto F, Cella M, Danieli C, and LanzavecchiaA. Dendritic cells use macropinocytosis and themannose receptor to concentrate macromoleculesin the major histocompatibility complex class IIcompartment: downregulation by cytokines andbacterial products. J Exp Med 1995.182: 389-400.

16. Spisek R, Brazova J, Rozkova D, Zapletalova K,Sediva A, and Bartunkova J. Maturation of dendri-tic cells by bacterial immunomodulators. Vaccine2004. 22: 2761-2768.

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INTRODUCTIONAccumulating evidence supports the hypothesis

that calcific aortic stenosis, conventionally viewed as a„degenerative“ process might be based on an activeinflammatory process (1, 2, 3). The sclerotic process inaortic stenosis presents many similarities with athero-sclerosis, including the presence of activated leuko-cytes in stenotic aortic valves (3). Moreover, an associ-ation between elevated inflammatory markers andadverse cardiovascular outcomes has been demonstra-ted in patients with aortic sclerosis, suggesting that in-flammation could be a common pathophysiologic me-chanism in both aortic sclerosis and atherosclerosis (4).

For a better definition of "vulnerable patient", se-veral markers of systemic inflammation were proposedas parameters that could predict the risk of future car-diovascular events (5).

In the current study, we applied this approach topatients with aortic sclerosis (AS). We examined the

serum levels of several inflammatory mediators - pro-and anti-inflammatory cytokines, matrix metallopro-teinases (MMPs), tissue inhibitor of metalloproteinase-1 (TIMP-1) and soluble intercellular adhesion mole-cule-1 (s-ICAM) in patients with aortic sclerosis. Theaim of the study was to see if elevated concentrationsof systemic inflammatory markers might be relevant inrelation with the progression of aortic valve calcifica-tion and also with the occurrence of acute coronaryevents.


Study populationFifty-one patients with aortic sclerosis (AS) were

consecutively registered and recruited at the Depart-ment of Cardiology, the Institute of CardiovascularDiseases „C.C. Iliescu“, Bucharest for the present study.The baseline characteristics are presented in Table 1.

ABSTRACTThe aim of the study was to evaluate several mediators of inflammation in patients with aortic sclerosisin relation to severity of cardiovascular disease. Serum level of cytokines, soluble intracellular adhesionmolecule 1, matrix metalloproteinase (MMP) 2 and 9 and their tissue inhibitor TIMP-1, were measuredby ELISA and MMPs activity by zymography in 51 aortic sclerosis patients. The increase in MMPs expres-sion positively correlated with their gelatinase activity; also there was a positive correlation betweenMMP-9 and TIMP-1 serum levels. Moreover, IL-6 concentration positively correlated with both serumlevel and activity of MMP-9. The level of IL-6 and IL-1Ra were higher in patients with a great burden ofatherosclerosis. Noteworthy, statistically significant higher levels of IL-6 were noticed for patients withcoronary artery disease. There was a significant increase in IL-6 serum level as well as a significantdecrease in IL-1Ra for patients with a history of myocardial infarction. A trend toward higher concentra-tion of inflammatory mediators was noticed in relation to the increase in severity of the aortic valve dis-ease. Our results support the hypothesis of an "inflammatory pattern" associated with AS pathology andsuggest the persistence of a chronic inflammation in patients who experienced acute coronary events.

SYSTEMIC INFLAMMATORY MARKERSIN PATIENTS WITH AORTIC SCLEROSIS

Mihaela Rugina1*, Iuliana Caras2, Ruxandra Jurcut1, Ciprian Jurcut3, Francisca Serbanescu2, Aurora Salageanu2 and Eduard Apetrei1

1Institute of Cardiovascular Diseases „C.C. Iliescu“, Bucharest, Romania; 2Infection and Immunity Laboratory, National Institute for Research and Development in Microbiology and Immunology „Cantacuzino“,

Bucharest, Romania and 3Central Emergency Military Hospital, Bucharest, Romania

Key words: aortic sclerosis; inflammation; cytokine; matrix metalloproteinase

*Corresponding author: M. Rugina, Institute of Cardiovascular Diseases „C.C.Iliescu“, 258 Fundeni Way, 022328 Bucharest,Romania, e-mail: [email protected]

Systemic inflammation and aortic sclerosis

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Twenty-one healthy subjects formed the control group(6 men and 15 women, mean age 28.6 ± 8.2 years).All the patients gave informed consent and the studywas conducted according to the guidelines approvedby the ethics committee of the Institute of Cardiovas-cular Diseases "C.C. Iliescu".

Exclusion criteria consisted of: presence of bicus-pid aortic valves (which could be a separate contribu-ting factor for thickening of aortic cusps), infectious,inflammatory or neoplasic known co-morbidities(which would be a confounding factor for the inflam-matory markers profile).

Clinical and echocardiography examinationStandard clinical examination was performed for

all patients during the first 48 hours of hospitalization.Each patient underwent an echocardiographicalassessment (HP Sonos 5500) for aortic valve morpho-logy (thickness, calcifications) and also Doppler para-meters (measurement of velocities and calculation ofmean and peak aortic gradient according to standardrecommendations). Aortic sclerosis was defined as anincreased echogenicity, thickening or calcification ofthe valve leaflets with a transaortic velocity below 2m/sec. A bilateral 2D carotid ultrasound examinationwas also performed in every patient for the assessmentof carotid intima-media thickness (IMT).

The patients were also assessed for the presence ofthe metabolic syndrome, according to the NCEP ATPIII criteria: 1) abdominal obesity (waist circumference>88 cm in women and >102 cm in men), 2) hypertri-glyceridemia ( 150 mg/dl), 3) low high density lipopro-tein (HDL) cholesterol (< 40 mg/dl in men and <50mg/dl in women), 4) high blood pressure ( 130/85mmHg), and 5) high fasting glucose ( 110 mg/dl).

Eighteen patients underwent coronary angiographyfor evaluation of coronary artery disease presence andextension. Four patients had single-vessel coronary

artery disease, eight patients had multi-vessel disease,and six had non-significant coronary stenosis.

Blood samplesBlood samples were obtained by venipuncture from

all the patients within 72h from the hospital admit-tance. For testing the inflammation markers, serum wasseparated and stored at -20oC until measuring.

Cytokines and other measurements Serum concentration of cytokines and other media-

tors was measured by enzyme-linked immunosorbentassay (ELISA) technique, using commercially availablekits as follows: IL-6 by high sensitivity ELISA (R&DSystems Europe Ltd, the lower limit of detection beingof 0.039 pg/ml); IL-1Ra, MMP-9, TIMP-1 and ICAM-1by Quantikine kits (R&D Systems, Abingdon, UK, thelower limit of detection of 3.9 pg/ml for IL-1Ra, 0.156ng/ml for MMP-9, 0.08 ng/ml for TIMP-1 and 0.35ng/ml for ICAM-1); IL-12 (p70) by BD OptEIA kits(Becton-Dickinson, San Diego, USA, the lower limit ofdetection of 7.8 pg/ml); IL-18 by MBL ELISA kit(Medical & Biological Laboratories, Japan, the lowerlimit of detection of 12.5 pg/ml).

Detection of MMPs activity by gel zymographyMMPs activity was tested by gelatin zymography.

Serum samples were diluted 1/100 in sample buffer[50mM Tris-HCl (pH=6.8), 10% (v/v) glycerol, 1% (w/v)sodium dodecyl sulfate (SDS), 0.5% (w/v) bromophe-nol blue] and separated in 8% SDS-PAGE gel polyme-rized with 1% (w/v) gelatin. Gels were removed fromglass plates and soaked for 30 min. in 2.5% Triton Xon a shaker followed by two brief washes in water.Thereafter gels were incubated overnight at 37oC inthe reaction buffer [50 mM Tris-HCl (pH 7.5), 0.2MNaCl, 5 mM CaCl2]. Gelatinolytic activity was visuali-zed by staining the gels with 0.25% Coomassie

Table 1. Baseline characteristics of the study patients

Brilliant Blue R-250 blue for 1 h on a shaker at roomtemperature. Destaining step was performed in glacialacetic acid: methanol: distilled water (1:3:6, v/v) solu-tion. Gels were scanned using a HP3690 scanner andMMP activity was quantified by densitometry. A sampleof fetal calf serum (FCS) was used as positive control and100% reference to determine relative bands intensity.

Statistical analysisResults were expressed as mean ± standard devia-

tion (SD). Continuous variables were compared bymeans of a two-sided Student's t-test or non-parametrictests. Spearman's correlation coefficient was used toassess the association between inflammatory markerslevels and clinical and echographic continuous vari-ables. Age correction was performed using a full facto-rial univariate linear model. A value of P < 0.05 wasconsidered statistically significant. Statistical analyseswere performed using SPSS Version 9.0 software.

RESULTS

Serum levels of inflammatory mediators in ASpatients vs. controls

When comparing the serum levels of inflammatorymarkers in patients and controls we noticed significanthigher levels of IL-6, MMP-9, TIMP-1 and sICAM-1 inAS patients as compared to healthy subjects (data notshown). To investigate the involvement of matrix metal-loproteinases, we assessed also the gelatinolytic acti-vity of MMP-2 and MMP-9 in patients and controls(Figure 1).

The two bands representing the latent form ofMMP-9 (92 kDa, upper band) and the latent form ofMMP-2 (72 kDa, lower band) were detected by gelzymography in the sera of all subjects, but the serumMMP-9 activities of aortic sclerosis patients were high-er than those of healthy controls. Densitometric analy-

sis in sera of 51 patients and 21 healthy controls indi-cated that the mean MMP-2 was comparable in thetwo groups while the MMP-9 activity for patients wasstatistically significant higher than controls. Furthermore,a good linear correlation was found between the den-sitometry units measured by zymogram and the res-pective concentrations of MMP-9 measured by immu-noassay in the sera of patients (r = 0.727, P <0.001)and controls (r = 0.572, P <0.01). A positive correla-tion was also noticed between measured TIMP-1 con-centrations and MMP-9 concentrations (r = 0.420, P<0.001) and MMP-9 activity (r = 0.373, P <0.05) instudied patients whilst no similar correlation wasfound in control group for these mediators.

Figure 1. Gelatinase activity of MMP-2 (72 kDa) and MMP-9 (92 kDa) in AS patients and controls. Sera from 51 ASpatients and 21 healthy controls were analyzed for theirMMP-2 and MMP-9 activities by gel zymography. A sampleof fetal calf serum (FCS) was used as positive control (lane1). The figure shows representative results for serum sam-ples from the two groups (lanes 2-5: controls, lanes 5-8: ASpatients).

However, it was demonstrated that advanced age isassociated with a hyperinflammatory state, referred toas 'inflamm-aging' (6). As there was a significant dif-ference in the age of control and patients groups, themeasured levels were age-adjusted (Table 2). As can beseen, after age correction, the values were still higher forIL-6, MMP-9 (level and activity), TIMP-1 and IL-18 andlower for IL-1Ra, in the patients group compared tocontrols although the differences were not statisticallysignificant (with the exception of sICAM-1).

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Table 2. Age-adjusted values of serum levels of inflammatory markers in AS patients and controls


13

Relation of systemic inflammatory markers to echo-graphic and angiographic measures of atherosclerosis

We wanted to see if there was any association bet-ween the increased values noticed for several inflam-mation mediators and the severity of atherosclerosisburden evaluated by carotid intima-media thickness(IMT). Indeed, we found higher age-adjusted values,although not statistically significant, for patients havinga "high" value of IMT (defined as an IMT 0.9 mm) ascompared to patients with IMT<0.9 mm for IL-6 (6 ±1.4 vs. 2.2 ± 1.7 pg/ml, P= 0.122), IL-1Ra (492.3 ±57.4 vs. 327 ± 67.5 pg/ml, P=0.083). Moreover, inthe case of IL-1Ra, a positive correlation was foundwith the measured values of IMT (r = 0.477, P < 0.05).

Next, we analyzed the serum concentrations ofinflammatory mediators in relation with the degree ofcoronary atherosclerosis evaluated by angiography.The noticed differences were at the level of IL-6 con-centration and MMP-9 activity, which were higher inthe subgroup of patients with multi-vessel disease com-pared to patients with less than two vessels affected(data not shown).

Relation of systemic inflammatory markers to car-diovascular risk factors, coronary artery disease andhistory of cardiovascular events

Generally, the age-adjusted values for systemic in-flammatory markers did not differ significantly bet-ween patients with different numbers of cardiovascularrisk factors. However, a slight increase of the serumlevels was noticed for patients with more than threerisk factors as compared to patients with less than threerisk factors for IL-6 (4.2 ± 0.6 pg/ml vs. 2.7 ± 1 pg/ml)and IL-1Ra (531.5 ± 44 pg/ml vs. 397.7 ± 67.3 pg/ml).

Diabetic patients had significantly higher levels ofIL-6 (Figure 2 ) and a higher MMP-9 activity (285.8 ±70 ng/ml in diabetic patients vs. 179.85 ± 40.9 ng/mlin non-diabetic patients). Also, patients with metabolicsyndrome had higher values of MMP-2 and MMP-9activities as well as higher values of IL-6 and IL-1 RAserum concentrations as compared to the otherpatients (data not shown).

Increased serum levels of inflammatory mediatorshave been reported to be correlated with the risk foracute coronary syndromes. In our study, 22 patientswere diagnosed with coronary artery disease (CAD). Forthese patients, the age-adjusted values of serum IL-6were significantly higher than for the other patients(Figure 3).

Among these CAD patients, 15 patients had a his-tory of myocardial infarction (MI). For this subgroup,the serum concentrations were significantly higher forIL-6 and lower for IL-1Ra as compared with no historyof infarction (Figure 4 A,B).

Figure 2. Serum concentration of IL-6 in AS patients in rela-tion to diabetes. Data represented as mean ± SD

Figure 3. Serum concentration of IL-6 in AS patients in rela-tion to CAD. Data represented as mean ± SD

For IL-6, serum level significantly correlated withthe severity of myocardial dysfunction as assessed bythe determination of ventricular ejection fraction (r =- 0.376, P < 0.05). No correlation was noticed in thecase of IL-1Ra.

Relation of systemic inflammatory markers to aor-tic valve disease

As the main objective of this study was to investi-gate the relevance of inflammatory markers in aorticsclerosis, we looked for any possible correlation bet-ween the serum levels of different mediators and theseverity of aortic valve disease, estimated by the de-gree of calcification, maximum aortic jet velocity andthe presence of aortic regurgitation.

We did not find a direct correlation between thedegree of calcification and any of the determinedinflammatory markers. However, a trend toward highervalues was noticed for IL-6 concentration with in-crease in the severity of aortic sclerosis (data notshown). Also patients with moderate and severe aortic

stenosis (maximum aortic jet velocity 2 m/s, n=10)had higher values of serum IL-6 than the others(5.1±1.4 pg/ml vs. 3.1±0.9 pg/ml) although the dif-ference was not statistically significant. Patients withaortic regurgitation (n=23) had significantly highervalues of total MMP activity (95.6±54.9 ng/ml vs.44.9±12.4 ng/ml, P <0.05) than patients without aor-tic regurgitation (n=14).

Figure 4. Serum concentration of IL-6 (A) and IL-1Ra (B) inAS patients in relation to the presence of a previous MI.Data represented as mean ± SD

DISCUSSION

Recently, it has been suggested that, similar to athe-rosclerosis, chronic inflammation may play a role inthe pathogenesis of aortic sclerosis (1, 2, 7, 8). As in-flammatory markers have been also considered a pre-dictor of future cardiovascular events such as acutecoronary syndromes, in this study we investigated apanel of inflammatory mediators in patients with aor-tic sclerosis.

In a first attempt to measure the inflammatorymarkers in patients vs. controls, we noticed statistical-ly significant increased values for IL-6, MMP-9, TIMP-1 and s-ICAM serum concentration in AS patients.

However, a major limitation of this study was the sig-nificant differences in the mean age of the two groups(62.9 ± 10.6 years in patients vs. 28.6 ± 8.2 years incontrol group) due to difficulty to find age-matchedsubjects without cardiovascular diseases. Therefore,we applied an age correction to the measured values.In these conditions, the differences between the twogroups were no longer significant, although higher val-ues were noticed for patients as compared to healthydonors for several parameters (Table 2). In particular,the level of IL-6 was greater in AS patients. IL-6 is animportant local and circulating marker of inflammationin cardiovascular tissues and increased levels werefound in patients with acute myocardial infarction andunstable angina (9, 10). In our study, the concentrationof IL-6 was significantly higher for patients with CADand also for patients with diabetes mellitus or meta-bolic syndrome. This is considered an important finding,as elevated levels of IL-6 were associated in literaturewith unfavorable short- and long-term prognoses inpatients with coronary artery disease (9).

Besides inflammatory cytokines, elevated levels ofcirculating soluble adhesion molecules have beenreported in patients with acute coronary syndromes(11, 12) and non-rheumatic aortic stenosis (13). In ourstudy we looked for serum levels of soluble intercellu-lar adhesion molecule 1 (sICAM-1) and found statisti-cally significant differences between patients and con-trols. Higher levels of circulating sICAM-1 in AS patientsmight be explained by activation of endothelium bycytokines that mediate the inflammatory response.

Matrix metalloproteinases (MMPs) represent a fa-mily of endopeptidases with proteolytic activity againstextracellular matrix components involved in variouspathological processes such as inflammation, tumormetastasis, respiratory diseases, myocardial injury, vas-cular aneurysms, and remodelling (14). It has beenshown that MMPs are upregulated in calcific AS andmight modulate matrix remodelling (15, 16). MMP-9(gelatinase B or 92-kDa type IV collagenase) was foundto be highly expressed in the vulnerable regions ofatherosclerotic plaques, and it has been suggested tobe causally involved in the remodelling processesassociated with atherogenesis and plaque rupture (14).In our study, we found positively correlated increasesin expression and activity of MMP-9 as well as inserum level of TIMP-1, a tissue inhibitor of MMPs inAS patients compared to controls, although the diffe-rences were not statistically significant after age cor-rection. Moreover, the IL-6 concentration positivelycorrelated to activity of MMP-9, suggesting an "inflam-matory pattern" of the investigated patients. As in thecase of IL-6 concentration, MMP-9 activity was signifi-cantly greater for patients with diabetes.

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A

B


15

It has been reported that the balance betweeninflammatory response by T-helper related cytokinesand anti-inflammatory response by T-helper-2 relatedcytokines is associated with coronary atherosclerosis(17). In our study, the age-adjusted serum levels of IL-6and IL-1Ra were higher in patients with a great burdenof atherosclerosis (defined by IMT 0.9 mm). Ourresults are in agreement with a previous report inwhich increased levels of IL-6 and IL-1Ra were foundin patients with stable angina pectoris, suggesting thatan increased inflammatory activity may play a role inchronic ischemic heart disease (18).

When the results were analyzed in relation withangiographic findings, we found higher serum levels ofIL-6 concentration and MMP-9 activity for patientswith multivessel disease, which suggest an "excessiveinflammation" pattern in patients with severe athero-sclerosis. However, our result does not support thehypothesis of a preferential involvement of Th-1 typeresponse in AS patients as reported by others in pa-tients with coronary artery disease (17, 19). A trendtoward higher concentration of serum IL-6 in relationto increase in the severity of aortic sclerosis was no-ticed. As we reported previously, values of IL-6 mightbe predictive for the presence of aortic valve calcifica-tions in patients with AS (20).

An interesting finding was the persistence ofincreased levels of IL-6 in patients who experienced anacute myocardial infarction. For these patients, age-adjusted serum concentration of IL-6 correlated signif-icantly with the severity of myocardial dysfunction asassessed by the determination of ventricular ejectionfraction. Recently, the role of interleukin-6 for LVremodelling and survival was re-examined in an exper-imental model using IL-6 knockout mice (21). Theauthors suggested that elevated levels of IL-6 in per-manent ischemia post-MI might reflect a response tothe extent of the underlying injury rather than beingdirectly involved in remodeling or heart failure. Ourresults might be explained by this hypothesis.

Through its capacity to bind to IL-1 receptor, thenaturally occurring antagonist IL-1 receptor antagonist(IL-1Ra) is considered to limit the potentially deleteri-ous effects of IL-1. The IL-1/IL-1Ra balance may deter-mine the severity of both acute and chronic inflamma-tion, and the relative absence of IL-1Ra is suggested toplay a role in the pathogenesis of some inflammatorydisorders, including atherosclerosis (22, 23). In agree-ment with other studies, which report increased ex-pression (23) or enhanced ex vivo synthesis of IL-1Ra(24) associated with cardiovascular events, we foundincreased levels of serum IL-1Ra in AS patients as com-pared to controls. However, we detected lower levelsof IL-1Ra (statistically significant after age correction)

in the group of patients with a history of MI, as com-pared to the other patients. In contrast to our results,early elevated levels of IL-1Ra have been reported inpatients with acute myocardial infarction (25). The dis-crepancy between our results and the cited study mightbe explained by the potential protective role of IL-1Raagainst deleterious effects of inflammatory cytokines. Itmay be presumed that in the case of post-MI patients,the level of IL-1Ra was insufficient to fully controlinflammation resulting in its long-term persistence.

In conclusion, in this study we demonstrated anassociation of several markers of systemic inflamma-tion with echocardiographic findings, suggesting an"inflammatory pattern" of aortic sclerosis patients. Asour study was not a prospective one and did not accu-mulate data sequentially with time, we could not in-vestigate the association between inflammation andadverse clinical outcomes in patients with aortic scle-rosis as suggested or the predictive value of studiedinflammation parameters (4). Despite this limitation andthe relatively small number of subjects, we demonstrat-ed that several inflammation mediators, such as IL-6,MMP-9, TIMP-1, IL-1Ra, IL-18 and sICAM-1 are pres-ent in higher concentration in sera of AS patients.Although the patients and control group were not age-matched, a relative increase in serum concentration ofinflammatory mediators was still noticed after age cor-rection in AS patients vs. healthy controls. In addition,our findings support the hypothesis of a chronicinflammatory "background" in patients with a historyof acute coronary events.

ACKNOWLEDGEMENTS

The authors thank Drs. Cristiana Matache and MariaStefanescu for help with gel zymography, Lucian Le-rescu and Catalin Tucureanu for help with statisticalanalysis and Dr. Jean-Marc Cavaillon for helpful dis-cussion and suggestions. This work was supported bya grant from the Romanian Ministry of Education andResearch (Program VIASAN, Grant 237/2004).

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4. Chandra HR, Goldstein JA, Choudhary N, O'NeillCS, George PB, Gangasani SR et al. Adverse out-come in aortic sclerosis is associated with coronaryartery disease and inflammation. J Am Coll Cardiol2004; 43:169-75

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6. Gomez CR, Boehmer ED, Kovacs EJ. The aginginnate immune system. Current Opinion inImmunology 2005;17:457-462

7. Jeevanantham V, Singh N, Izuora K, D'Souza JP,His DH. Correlations of high-sensitivity C-reactiveprotein and calcific aortic valve disease. MayoClinic Proc 2007;82(2):171-174

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9. Ito T, Ikeda U. Inflammatory cytokines and cardio-vascular disease. Curr Drug Targets Inflamm Allergy2003; 2:257-65

10.Rallidis LS, Zolindaki MG, Pentzeridis PC,Poulopoulos KP, Velissaridou AH, Apostolou TS.Raised concentrations of macrophage colony sti-mulating factor in severe unstable angina beyondthe acute phase are strongly predictive of long termoutcome. Heart 2004; 90:25-29

11.Murphy RT, Foley JB, Crean P, Walsh MJ. Re-ciprocal activation of leukocyte-endothelial adhe-sion molecules in acute coronary syndromes. Int JCardiol 2003;90:247-52

12.Rallidis LS, Gika HI, Zolindaki MG, Xydas TA,Paravolidakis KE, Velissaridou AH. Usefulness ofelevated levels of soluble vascular cell adhesionmolecule-1 in predicting in-hospital prognosis inpatients with unstable angina pectoris. Am JCardiol 2003;92:1195-97

13.Shahi CN, Ghaisas NK, Goggins M, Foley B, CreanP, Kelleher D et al. Elevated levels of circulatingsoluble adhesion molecules in patients with non-rheumatic aortic stenosis. Am J Cardiol 1997;79:980-82

14.Blankenberg S, Rupprecht HJ, Poirier O, Bickel C,Smieja M, Hafner G et al. Plasma concentrationsand genetic variation of matrix metalloproteinase 9and prognosis of patients with cardiovascular dis-ease. Circulation 2003; 107:1579-85

15.Kadena JJ, Dempflea C-E, Grobholz R, Trana H-T,Kýlýç R, Sarýkoç A et al. Interleukin-1 beta pro-motes matrix metalloproteinase expression and cellproliferation in calcific aortic valve stenosis.Atherosclerosis 2003; 170:205-11

16.Edep ME, Shirani J, Wolf P, Brown DL. Matrix me-talloproteinase expression in nonrheumatic aorticstenosis. Cardiovasc Pathol 2000; 9:281-6.

17.Yamashita H, Shimada K, Seki E, Mokuno H,Daida H. Concentrations of interleukins, interfer-on, and C-reactive protein in stable and unstableangina pectoris. Am J Cardiol 2003; 91:133-36

18.Gabriel AS, Ahnve S, Wretlind B, Martinsson A.IL-6 and IL-1 receptor antagonist in stable angina pec-toris and relation of IL-6 to clinical findings in acutemyocardial infarction. J Intern Med 2000; 248: 61-66

19.Fernandes JL, Mamoni RL, Orford JL, Garcia C,Selwin AP, Coelho OR et al. Increased Th1 activityin patients with coronary artery disease. Cytokine2004; 26:131-137

20.E Apetrei, M Rugina, Jurcut R, Salageanu A, JurcutC, Caras I et al. Predictors of calcification develop-ment in patients with aortic sclerosis [abstract]. EurHeart J 2005 Suppl:P1230

21.Fuchs M, Hilfiker A, Kaminski K, Hilfiker-KleinerD, Guener Z, Klein G et al. Role of interleukin-6for LV remodeling and survival after experimentalmyocardial infarction FASEB J 2003;17:2118-20

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25.Patti G, D'Ambrosio A, Mega S, Giorgi G, ZardiEM, Zardi DM et al. Early interleukin-1 receptorantagonist elevation in patients with acute myocar-dial infarction. J Am Coll Cardiol. 2004; 43:35-8

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INTRODUCTION

Vaccines are considered to be one of most success-ful medical interventions against infectious disease.Many significant obstacles remain such as improvingvaccines against yet undefeated pathogens, developingnew ones against diseases for which no vaccines yetexist, responding rapidly to newly emerging pathogensand successful intervention in chronic diseases inwhich ongoing immune responses are insufficient [1,2]. The goal of vaccination is the generation of a strongimmune response to the administered antigen able toprovide long-term protection against infection. Purerecombinant or synthetic antigens used in modern dayvaccines are generally far less immunogenic than olderstyle live or killed whole organism vaccines so modernvaccines often requires the addition of an adjuvant.Adjuvant mechanisms of action include: increasing thebiological or immunologic half-life of vaccine anti-gens; improving antigen delivery to antigen-presentingcells (APCs), as well as antigen processing and presen-tation by the APCs; inducing the production of regula-tory cytokines that favor the development of T-helpertype 1 (Th1) or type 2 (Th2) immune responses to vac-cine antigens [3, 4]. However, despite extensive eva-luation over many years [5], aluminum compoundssuch as aluminum hydroxide and aluminum phos-phate are the most widely used adjuvants in human

vaccines. Unfortunately they do not fulfill the optimalrequirements for a potent immune adjuvant. Limita-tions of aluminum adjuvants include local reactions,augmentation of IgE antibody response, ineffectivenessfor some antigens and inability to elicit cell-mediatedimmune responses especially cytotoxic T-cell respons-es [6]. Therefore, development of more effective adju-vant capable to replace alum is imperiously necessary.

CANTASTIM is a second generation bacterial im-munomodulator produced in our institute, obtained byextraction from a strain of Pseudomonas aeruginosa.The product is a mixture of bacterial components,comprised mainly of lipids (>80%), lipoproteins, lipo-peptides, muramylpeptides and neutral sugars. Experi-mental studies in vivo and in vitro demonstrated thatCS has non-specific protective effect against bacterialinfections and endotoxin shock, activates monocytes /macrophages (enhancement of bactericidal activity,induction of cytokines and other mediators, such asnitric oxide), T lymphocytes and NK cells [7, 8]. In thepresent study, CS was investigated as an adjuvant for amodel protein vaccine, TT.


ReagentsTetanus toxoid was a kind gift from S. Durbaca from

Tetanus Laboratory, NIRDMI, Bucharest, Romania.

ABSTRACTAluminum compounds have been used as adjuvants in practical vaccination for more than 60 years toinduce an early, an efficient and a long lasting protective immunity. Nowadays they are the most widelyused adjuvants in both veterinary and human vaccines. Unfortunately these adjuvants do not only causeundesirable side effects, but often induce T-helper type 2 (Th2)-biased responses. In this study we inves-tigated the ability of the bacterial product CANTASTIM (CS) to augment the immune responses to a modelantigen, tetanus toxoid (TT). Immunization of mice with TT+CS elicited higher anti-TT IgG antibody lev-els as compared to mice that received TT alone. Moreover, treatment with TT+CS resulted in a lowerIgG1/IgG2a ratio and a stronger in vitro IFN-γ synthesis by splenocytes and T cells cocultured withmacrophages. These data suggest that CS can be used to enhance the magnitude of the immune responseand to skew it towards the Th1 type.

ADJUVANT PROPERTIES OF BACTERIAL PRODUCT CANTASTIM

Iuliana Francisca Anghelache*, Iuliana Caras* and Aurora Salageanu

Infection and Immunity Laboratory, "Cantacuzino" National Institute of Research and Development for Microbiology and Immunology (NIRDMI), Bucharest, Romania

Key words: CANTASTIM, adjuvant, tetanus toxoid, vaccines

*Corresponding author: E-mail: [email protected] authors contributed equally to the paper

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AnimalsThe mice used in the experiments (female BALB/c

mice at the age of 6-8 weeks) have been housed andmaintained in accordance with institutional guidelinesunder approved authority protocols and checked onregular intervals by veterinarians and handled by qua-lified animal care takers within the authorized animalfacility of our institute. To ensure that the suffering ofthe animals was kept to a minimum, all manipulationswere performed in accordance with the proceduresalready approved by the local ethical committee andwith the guidelines and regulations of the Romaniananimal protection law.

Immunization of miceGroups of ten mice were immunized subcuta-

neously (s.c.) with 10 µg TT in PBS, either alone or to-gether with 1mg CS. These concentrations were cho-sen based on preliminary experiments (CS dose) andliterature data (TT dose). On days 7, 14, 21, 35 and 42,mice from each group were boosted with the same for-mulation as was used at prime. Animals were bled byretro-orbital puncture at regular time intervals afterimmunization. Serum samples were stored at -70oCuntil assayed.

Spleen Cell Culture Three days after the last immunization, mice were

sacrificed, spleens were aseptically removed, and asingle cell suspension was prepared in RPMI 1640medium (Sigma) supplemented with 10% fetal calfserum (FCS, InVitrogen), 1 mM L-glutamine, 100 U/mLpenicillin, 100 µg/mL streptomycin and 10-5M ß-mer-captoethanol. Aliquots of the cells were cultured at 1 ×106 cells/well (COSTAR® 96-well round bottom plates,Corning Inc.), stimulated with TT (1 µg/mL) and incu-bated for 7 days, at 37oC in 5% CO2 at approximately95% humidity. Unstimulated cells served as negativecontrol. After incubation, the supernatants were reco-vered and stored at -20oC until assayed for IFN-γ.

Macrophage-T cell coculturePeritoneal exudate cells were obtained by two

rounds of peritoneal lavage with 5 mL of PBS. Cellswere collected by centrifugation, washed twice, andthen resuspended in complete medium (RPMI 1640medium supplemented with 10% FCS, 1 mM L-gluta-mine, 100 U/mL penicillin, 100 µg/mL streptomycin).To obtain macrophages, peritoneal exudate cells wereplastic adhered by incubating cells at 2x105 cells/well(COSTAR® 96-well flat bottom plates, Corning Inc.) for2 h in 5% CO2 at 37°C. After 2 h nonadherent cells wereremoved by gentle aspiration and washing with PBSand adherent macrophages were further cultured incomplete medium.

Splenocyte suspension was enriched for T cellsusing T cell enrichment columns (R&D Systems).Enriched T cells were added to adherent peritonealmacrophages at a ratio of 3:1 and cocultured cellswere stimulated with TT (1 µg/mL). Cell supernatantswere harvested after 7 days of culture and stored at-20°C until assayed for IFN-γ.

Measurement of serum total IgG and subtypestiters by ELISA

Anti-TT antibodies in mouse serum were detectedand quantified by end-point titration using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-wellmicro-titer plates (Nunc) were coated with 1 µg TT/wellin 0.1M sodium carbonate buffer (pH=9.5) and incu-bated overnight at room temperature (RT). The unre-acted sites were blocked with 100 µL/well of 1%bovine serum albumin (BSA) in PBS for 1h at RT. Plateswere washed three times with 0.05% Tween-80 in PBSfollowed by the addition of test samples in duplicates.Serum obtained from unimmunized mice served asnegative control. Serum samples were serially dilutedin 1% BSA and 0.05% Tween-80 in PBS, added in 50µL aliquots per well and incubated for 1h and 30 minat 37oC. After washing three times, 50 µL/well of alka-line-phosphatase conjugated goat anti-mouse IgG(whole molecule) (Sigma), alkaline-phosphatase conju-gated anti-mouse IgG2a (BD Biosciences Pharmingen)and horseradish peroxidase conjugated anti-mouseIgG1 (BD Biosciences Pharmingen), respectively, wereadded at appropriate dilutions to different assay plates.Following 1h incubation at 37oC and three washes,100 µL of para-Nitrophenylphosphate (pNPP) (0.36mg/mL) and Orthophenylenediamine (OPD) substrate(0.4 mg/mL), respectively, were added to the wells.After color development, the reaction was stopped byaddition of 2N NaOH and 2N H2SO4, respectively(100 µL/well). The absorbance was measured at 405 and490 nm, respectively, in an ELISA microplate reader.

Each mouse serum sample was tested separately forantibody titer.

End-point dilution titers for total IgG as well asIgG1 and IgG2a subtypes were defined as the highestserum dilution that resulted in an absorbance valuehigher than a cut-off value of 0.04.

IFN-γγ ELISA assay Levels of IFN-γ in culture supernatants were meas-

ured using the Quantikine ELISA kit according to themanufacturer's instructions (R&D Systems). The resultswere expressed as mean IFN-γ concentrations ± thestandard deviation (SD), after extrapolation from astandard curve prepared with standard cytokine.

Adjuvant properties of bacterial product CANTASTIM

19

Statistical AnalysisData were analyzed using a paired Student's t test.Results were considered significant only if p < 0.05.

RESULTS

Serum antibody responses after immunizationwith TT plus CS

To examine the ability of CS to enhance antibodytiters to TT, serum from immunized mice was collec-ted and tested one week post boost. As shown inFigure 1, mice immunized with TT+CS exhibited sta-tistically significant higher IgG titers to TT than thegroup immunized only with TT starting with 1 weekafter the 2nd immunization (p=0.038). The differencein antibody titers increased progressively after the 4th,5th and 6th immunization (p=8.8 x 10-8, p=6.4 x 10-5

and p=4.2 x 10-5, respectively).As expected, the level of anti-TT antibody in unim-

munized mice was under the detection limit of themethod (data not shown).

Subtypes of serum antibody responses after immu-nization with TT plus CS

The amount of IgG1 and IgG2a raised against TTwas measured by ELISA 1 week after the 4th, 5th and6th immunization (Figure 2). The titers of IgG1 andIgG2a in mice immunized with TT+CS were statisti-cally significant higher than those in mice immunizedwith TT alone starting with 1 week after the 4th immu-nization (p=4.97 x 10-5 and p=0.005, respectively).

To analyze the Th1/Th2 balance the IgG1/IgG2aratio was determined. Higher ratios of IgG1/IgG2aindicate a dominant Th2-type while lower ratios a Th1-type immune response [9]. The IgG1/IgG2a ratio was

significantly lower in the group of mice immunizedwith TT+CS as compared to the group immunizedwith TT alone (table 1). These results suggested that CSfavored a shift towards a Th1-type immune response.

Effect of CS on IFN-γγ ProductionTypically, the secretion of IFN-γ is indicative of a

Th1-type of immune responses. To evaluate the influ-ence of CS on IFN-γ secretion, splenocytes from immu-nized mice were stimulated as described in Materialsand Methods and IFN-γ concentration was measuredin cell culture supernatants by ELISA. Our resultsshowed that splenocytes of mice immunized withTT+CS produced a higher but not statistically signifi-cant level of IFN-γ than those immunized with TTalone (5982±4008 versus 3002±1816 pg/ml). It isknown that a number of different cell types, includingTh, CTL, and NK cells, are capable of producing IFN-γin response to stimulation. Therefore, to study theinfluence of CS on the IFN-γ production by T cells,splenocyte suspension from immunized mice wasenriched for T cells. In one coculture experiment wenoticed that cells from mice that received CS producedhigher concentration of IFN-γ in response to TT com-pared with mice that were immunized with TT alone(695 versus 355 pg/mL).

DISCUSSION

Adjuvants are commonly used in vaccines to en-hance immune responses. Development of the appro-priate type of immune response is essential for suc-cessful immunization. Strong cell-mediated immunitythat is associated with a Th1 type immune response isthought to be essential for the control of intracellular

Figure 1 - Total anti-TT IgG in sera after immunization. Ten Balb/c mice per group wereimmunized s.c. with TT alone or with TT+CS. Mice were bled 1 week after the 2nd immu-nization (week 2), 4th immunization (week 4), 5th immunization (week 5) and 6th immu-nization (week 6). The antibody titers of serum specimens were measured by ELISA. Eachbar represents the mean value of antibody titers ± standard deviation of ten mice per group.*p<0.05; **p<0.001, as compared to TT alone (Student's two-tailed t-test)

20

ANGHELACHE et al.

pathogens whereas strong humoral immunity, whichcan be found with both Th1 and Th2 type immuneresponses, appears to be essential for the control ofextracellular pathogens. Alum is currently the onlyadjuvant licensed for human use in most countries.Although it is a very safe and inexpensive adjuvant,there are some inherent limitations associated with itsuse like the inability to induce strong Th1-typeimmune responses (essential against intracellularpathogens), the necessity of a multi-injection scheduleand a short shelf life. The need to find alternate adju-vants has become even more pronounced afteradvancements in the field of DNA and recombinantprotein technologies [10, 11]. Adjuvants such as mo-nophosphoryl lipid A (MPL), the saponin derivativeQS-21, as well as combinations of these adjuvants arein late stage clinical testing. MPL is licensed in somecountries as part of the allergy vaccine Pollinex Quattrowhile others such as incomplete Freund's adjuvant,cytokine/growth factors, CpG motifs, muramyl tripep-tide and liposomes are in clinical development and

many more are at the pre-clinical testing stage [12].Numerous studies have demonstrated that cyto-

kines produced by APCs and NK cells during theinnate immune response are capable of affecting de-velopment of acquired immune responses. Interleukin(IL)-12 is critical for the development of Th1 cells thatare involved in cell-mediated immunity. IL-12 can alsocontribute to the induction of IFN-γ and activation ofCTL [13]. IFN-γ is an important B cell switch factor forthe induction of antigen-specific IgG2a-secreting Bcells [14].

In this study we investigated the potential of CS tomodulate the immune response elicited by immuniza-tion with TT antigen. We showed that administrationof CS concomitantly with TT induced statistically sig-nificant higher titers of TT-specific total IgG thanadministration of TT alone after two immunizations.The antibody titers increased progressively with thenumber of immunizations for both groups. However,the titers were statistically significant higher for themice immunized with TT + CS after each booster

Figure 2 - Titers of anti-TT IgG subclass antibodies in sera of immunized mice. Ten Balb/cmice per group were immunized s.c. with TT alone or with TT+CS. Mice were bled 1 weekafter the 4th immunization (week 4), 5th immunization (week 5) and 6th immunization(week 6). IgG1 (A) and IgG2a (B) titers were measured by ELISA. Each bar represents themean value of antibody titers ± standard deviation of ten mice per group. *p<0.05;**p<0.001, as compared to TT alone (Student's two-tailed t-test).

Adjuvant properties of bacterial product CANTASTIM

21

immunization. Isotyping analysis of immunoglobulinsin serum specimens revealed that CS augmented thetiters of both IgG1 and IgG2a. Importantly, theIgG1/IgG2a ratio was lower in the group of mice thatreceived CS suggesting a shift to a Th1-dominantimmune response. In addition, in this study weshowed that cells isolated from mice immunized withTT+CS produced more IFN-γ compared with mice thatreceived only TT. The augmentation of IFN-γ secretionfurther emphasizes the dominance of the Th1 over Th2type of immune response. In previous studies, wenoticed a slight increase in the level of IL-12 upon invitro stimulation of whole blood with CS [8]. We havealso shown that stimulation of whole blood cultureswith CS increased both the frequency and the expres-sion of CD69 on the surface of T lymphocytes and NKcells [7]. Thus, CS may act directly on NK cells to pro-duce IFN-γ or activate monocytes to produce IL-12, orboth.

In summary, our data indicate that CS has a potentadjuvant effect for TT antigen when delivered subcuta-neously to mice. Also, immunization of mice with TTin the presence of CS induced lower IgG1/IgG2a ratioand an increased IFN-γ production indicating a Th1-biased immune response. The ability to induce apotent Th1-type immune response is of considerableimportance, because for many pathogens, cell-mediat-ed immunity and not the presence of antibody is cor-related with protection. In conclusion, our resultsdemonstrate that CS could be an attractive candidateto be used as adjuvant.

AcknowledgementsThe present study was supported by BIOTECH Pro-

gram for Research of Romanian Ministry of Educationand Research.

The skillful technical assistance of Camelia Tabarta,Rodica Tudorache and Chioseolu Florica is gratefullyacknowledged.

REFERENCES

1. Pashine A, Valiante NM, Ulmer JB. Targeting theinnate immune response with improved vaccineadjuvants. Nat Med 2005. 11(4):S63-68

2. Wack A, Rappuoli R. Vaccinology at the beginningof the 21st century. Curr Opin Immunol 2005.17(4):411-418

3. Vogel FR. Improving vaccine performance with adju-vants. Clin Infect Dis 2000. 30(Suppl3):S266-270

4. Petrovsky N, Aguilar JC. Vaccine adjuvants: currentstate and future trends. Immunol Cell Biol 2004.82(5):488-496

5. Edelman R. The development and use of vaccineadjuvants. Mol Biotechnol 2002. 21(2):129-148

6. Gupta RK. Aluminum compounds as vaccine adju-vants. Adv Drug Deliv Rev 1998. 32(3):155-172

7. Caras I, Serbanescu IF., Grigorescu A and Sala-geanu A. Experimental studies on bacterial productCANTASTIM derived from Pseudomonas aerugi-nosa. VII. Activation of immune cells of healthycontrols and cancer patients. Roum Arch MicrobiolImmunol 2005. 64(1-4): 5-10.

8. Lerescu L, Tucureanu C, Caras I and Salageanu A.Cytokine Profiling by Multiplex Immunoassay as anEffective Approach to Assess ImmunomodulatoryActivity of Bacterial Product CANTASTIM. RomArch Microbiol Immunol. 2006. 1-2:53-58

9. Mussalem JS, Vasconcelos JR, Squaiella CC, Ana-nias RZ, Braga EG, Rodrigues MM, Longo-MaugériIM. Adjuvant effect of the Propionibacterium acnesand its purified soluble polysaccharide on the immuni-zation with plasmidial DNA containing a Trypanosomacruzi gene. Microbiol Immunol 2006. 50(4):253-263

10. Weeratna RD, McCluskie MJ, Xu Y, Davis HL.CpG DNA induces stronger immune responseswith less toxicity than other adjuvants. Vaccine2000. 18(17):1755-1762

11. Diwan M, Tafaghodi M, Samuel J. Enhancement ofimmune responses by co-delivery of a CpG oligo-deoxynucleotide and tetanus toxoid in biodegra-dable nanospheres. J Control Release 2002. 85(1-3):247-262

12. Brennan FR, Dougan G. Non-clinical safety evalu-ation of novel vaccines and adjuvants: new products,new strategies. Vaccine 2005. 23(24):3210-3222

13. Wagner TL, Ahonen CL, Couture AM, Gibson SJ,Miller RL, Smith RM, Reiter MJ, Vasilakos JP, TomaiMA. Modulation of TH1 and TH2 cytokine produc-tion with the immune response modifiers, R-848 andimiquimod. Cell Immunol 1999. 191(1):10-19

14. Ioannou XP, Gomis SM, Karvonen B, Hecker R,Babiuk LA, van Drunen Littel-van den Hurk S.CpG-containing oligodeoxynucleotides, in combi-nation with conventional adjuvants, enhance themagnitude and change the bias of the immuneresponses to a herpesvirus glycoprotein. Vaccine2002. 21(1-2):127-137

Table 1IgG1/IgG2a ratio in sera of immunized mice

Ten Balb/c mice per group were immunized s.c. with TTalone or with TT+CS. Mice were bled 1 week after the 6thimmunization (week 6). The numbers represent the meanvalue of IgG1/IgG2a ratio (ten mice per group); p=0.006

22

INTRODUCTION

Nucleoside monophosphate kinases (NMPk) cata-lyze the reversible transfer of the terminal phosphorylgroup from a nucleoside triphosphate (in most casesATP) to nucleoside monophosphate. In mammaliantissues at least four distinct NMP kinases have beenidentified biochemically: adenylate kinase (AMP kina-se), guanylate kinase (GMP kinase), thymidylate kinase(dTMP kinase) and pyrimidin nucleoside monophos-phate kinase (CMP-UMP kinase).

Whereas adenylate kinase from various sources havebeen studied extensively over many years, both bybiochemical and biophysical methods, much less isknown about guanylate kinase (ATP:GMP phospho-transferase, EC 2.7.4.8) [1].

Guanylate kinase (GMPK) catalyzes the first step inreversible phosphorylation of either GMP to GDP ordGMP to dGDP and is an essential enzyme in nucleo-tide metabolic pathways [2].

Guanylate kinase also serves to potentiate antiviraldrugs activity in virus infected cells. For example acti-vation of the guanosine analogs acyclovir and ganci-clovir after an initial phosphorylation step by the her-pes viral thymidine kinase is carried out by guanylatekinase [3-4].

In the present work we tried to characterize GMPK,studying its expression, activity and kinetics, usingpolymerase chain reaction (PCR) and culture assays.


Bacterial strainsWe use the strains Streptococcus pneumoniae

1131 stereotype 4 from the Cantacuzino Institute's col-lection of the National Center Reference for Strepto-coccus and Pseudomonas aeruginosa PA01.

Cloning and ExpressionThe genomic DNA was purified by wizard Geno-

mic DNA Purification kit (Promega). The genes forguanylate kinase were amplified by PCR from the twostrains. The PCR reaction is was performed with AppliedBiosystems Termocycler, using the program: 5 min at94°C, 25 cycles (30 sec. at 94°C, 45 sec. at 60°C and1 min. at 72°C), 7 min. at 72°C.

The primers used for PCR were:P. aeruginosa

Upp: 5' GGG AAT TCC ATA TGT CCG GCA CCCTG 3' (26 mer)

Low: 5' CGG GAT CCT CAG GCG AGC AGACGA CC 3' (26 mer)S. pneumoniae

Upp: 5' GGG AAT TCC ATA TGG CAG ACC GAGGC 3' (26 mer)

Low: 5' CGC GGA TCC TCA TCG GGT AGT TGGAG 3' (26 mer)

The PCR products were cloned into pET 24 andpET 28 plasmids (Novagen) between Nde I and Bam

ABSTRACTGuanylate kinase is a member of the nucleoside monophosphate (NMP) kinase family, a family ofenzymes that despite having a low primary structure identity share a similar fold, which consists of threestructurally distinct regions termed the CORE, LID, and NMP-binding regions. Guanylate kinase (GMPK)is an essential enzyme for the biosynthesis of GTP and dGTP by catalyzing the phosphoryl transfer fromATP to (d)GMP resulting in ADP and (d)GDP. Despite the similar fold of the monomer there is an impor-tant difference between GMPKs from prokaryotes and eukaryotes: eukaryotes GMPK are monomers whileprokaryotes GMPK are dimmers, tetramers or hexamers. For this reason bacterial GMPKs are possible tar-gets for new antibacterial drugs. Finding new targets for antibacterial therapies is a prior subject in today'smedical research. The purpose of this work was to characterize guanylate kinases from both gram posi-tive and gram negative pathogenic bacteria. We started with GMPK from Enterococcus faecalis as grampositive microorganism and Pseudomonas aeruginosa as gram negative representative.

CHARACTERIZATION OF GUANYLATE KINASE FROM GRAM POSITIVEAND GRAM NEGATIVE MICROORGANISMS; PRELIMINARY RESULTS

Ana-Maria Ruxandra Eftimie, Florina Toma, Adriana-Zoe Costache and Nadia Bucurenci

(N.I.R.D.M.I „Cantacuzino“ - Bucharest, Romania

Key words: guanylate kinase, DNA cloning, guanosine 5`-monophosphate, ATP regeneration, acyelovir, ganciclovir

Characterization of guanylate kinase from gram positive and gram negative microorganisms; preliminary results

23

HI restriction sites for P. aeruginosa gmk and betweenNde I and XhoI restriction sites for S. pneumoniae gmk.The DNA recombinants were transformed into the E.coli strain BL 15. Bacteria were grown at 37°C on 2YTmedium supplemented with 30 µg/ml Kanamycin and30 µg/ml Chloramphenicol. In order to induce GMPKexpression, 1mM isopropyl-ß-D-thiogalactosidase (IPTG)was added (when the optical density at 600nmreached 1.3). At 3 hours after induction bacteria wereharvested by centrifugation.

Protein Purification and CharacterizationThe bacterial pellet was resuspended in specific

buffer (TRIS-HCl 50 mM pH=7.4 for pET 24, andmonosodic phosphate 50 mM pH=8, NaCl 300 mMand imidazol 10 mM for pET28) and kept at -20°C [5].

The bacterial suspension was sonicated for 12 mi-nutes in a pulse mode at 4°C. The resulting lysate wascentrifuged for 45 min at 10 000 rpm. The expressionwas verified by SDS-PAGE. The supernatant was mea-sured and loaded onto a Ni-NTA gel column in case ofthe protein cloned in pET 28, and onto a DEAE Sepha-rose gel column in case of the protein cloned in pET24. The next step is concentration on a 10 KDa Filtronuntil we reach 20 mg/ml concentration. After this weload onto a column of Sephacryl S-200.

Fractions from the gel filtration column were mo-nitored by A280 in case of pET 24, and by SDS-PAGE incase of pET 28. Specific activity was also determined.

Steady-state KineticsThis method permits determination of the enzy-

matic activity by following the decrease of NADH con-centration at 340 nm.

RESULTS

1. By PCR, out of S. pneumoniae, the colonies 4 and12-16 were positive, whereas the rest are negative(Fig. 1).

2. By expression we've demonstrated that our ampli-cons are at the right molecular weight, and that theproteins are very well expressed (Fig. 2).In case of using vector pET 24 the purification ismade in two steps, first step consists in loading ontoa column of DEAE Sephacel, then follows a concen-tration, and the second step consists in loading ontoa column of Sephacryl S-200 for final purification(Fig. 3 and 4).

3. By steady-state kinetics we have obtain the graphicsfor S. pneumoniae and P. aeruginosa (Fig. 5 and 6)and (Fig. 7 and 8).

Fig 1 - Gel electrophoresis (1%) of PCR products for gmkgene in Streptococcus pneumoniae strain. Line 1 (positivecontrol, c1+ and c2+, for gmk amplicon, and negativecontrol, c-), Lines 2›16 colonies. Lines 4, 12,13, 14, 15, 16positive colonies

Fig 2 - SDS-PAGE 12, 5%, ET-total extract, EI-insolubilextract, ES- solubil extract, MW- molecular weight marker200 kDa, 116 kDa, 97.4 kDa, 66 kDa, 45 kDa, 31 kDa,21.5 kDa, 14.5 kDa

Fig 3 - Purification example for Pseudomonas aeruginosa,a pathogenic gram negative bacterium, and wich was loadedonto a DEAE Sepharose gel

Fig 4 - Purification example for Pseudomonas aeruginosa,a pathogenic gram negative bacterium, and wich wasloaded on a Sephacryl S200 column

Fig 5 - GMPK Streptococcus pneumoniae kinetics for pH=6

Fig 6 - GMPK Streptococcus pneumoniae kinetics for pH=8

Fig 7 - GMPK, Pseudomonas aeruginosa kinetics for pH=6

Fig 8 - GMPK, Pseudomonas aeruginosa kinetics for pH=7.4

DISCUSSION

The results obtained in the studies of guanylatekinases from pathogen microorganisms will permit toelaborate new models to conceive specific inhibitorsas antibacterial drugs.

The ability of guanylate kinase to catalyze the trans-fer of phosporyl group from ATP to GMP to yield GDPis important physiologically. Being a component of themetabolic pathway of GTP and dGTP formation,GMPK plays a vital role in DNA and RNA synthesis (6).

Additionally, the recycling of the second messengercGMP and the overall concentrations of GTP, which isinvolved in the regulation of numerous pathways, isdependent on GMPK activity.

The medical importance of GMPK is the phospho-rylation of guanosine analog prodrugs (7).

24

EFTIMIE et al.

Characterization of guanylate kinase from gram positive and gram negative microorganisms; preliminary results

25

We tested the GMP and ATP as substrates for GMPKby kinetics in the forward reaction. It is noted that thespecific activity of this enzyme was labile during sto-rage. One way to check the consistency of the kineticparameters is to measure the equilibrium constant ofthe reaction. Partial substrate inhibition by excess ofGMP has been observed in several cases.

The kinetics revealed that Vmax maximum of ourenzymes is at pH 6.0.

We will continue with our studies with GMPK fromother pathogens trying to find eventual differencesbetween enzymes from gram negative and gram posi-tive organisms. And last but not least we will try to findinhibitors for bacterial GMPK among natural or modi-fied nucleotides.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the skilful tech-nical assistance of FERARU ECATERINA and ENACHEVASILICA.

REFERENCES

1. BESSMAN, M.J. and VAN BIBBER, M.J. - Purifi-cation and Properties of Guanylate Kinase fromEscherichia coli. Biochem. Biophys. Res. Commun.,1 (101), 1959

2. MIECH. R.P. & PARKS. R.E. Jr. - Adenosine triphos-phate, guanosine monophosphate phosphotrans-ferase. Partial purification and substrate specificity.J. Biol. Chem., 240 (351-357), 1965

3. OESCHGER, M.P& BESSMANN. M.J. - Purificationand properties of guanylate kinase from Escherichiacoli. J. Biol. Chem., 241 (5452-5460), 1966

4. N.SEKULIC, L. SHUVALOVA, O. SPANGENBERG,M. KONRAD, A. LAVIE. - Structural characteriza-tion of the closed conformation of mouse guanylatekinase. J. Biol. Chem., 277 (30236-30243), 2002

5. PACE, C.N. - Structural and Functional Roles ofTyrosine 78 of Yeast Guanylate Kinase. MethodsEnzymol.,131 (266-280), 1986

6. PETROS, A.M., MUELLER, L. and KOPPLE, K.D. -Biochemisty., 29 (10041-10048), 1990

7. YPHANTIS, D.A., Biochemisty., 3 (297),1964

1. INTRODUCTION

As knowledge has accumulated on the blood-transmitted pathogenic agents, the contact with bio-logical fluids (blood, plasma, saliva, etc) from appar-ently healthy individuals has started to be regarded asa real professional risk for medical and sanitary staff.From this viewpoint, dentistry ranges among the spe-cialties having a high professional risk.

Theoretically, exposure to a contaminated bio-logical specimen may have as a consequence trans-mission of infection from patient to dentist, from den-tist to patient and from patient to patient via inade-quately decontaminated and sterilized dental equip-ment. Nevertheless, studies have shown that trans-mission from dentist to patient is quite rare as not all theconditions involved in transmission are met: presenceof viremia, presence of a lesion that causes bleedingand the existence of conditions that enable access ofthe infected dentist's blood to the patient's wound ormucous membranes (1). A more frequent occurrenceseems to be transmission from patient to dentist orcross-contamination among patients.

It is common knowledge that infection risk isinfluenced by a number of factors, such as: the infec-tion source (incidence/prevalence in population, thesize of the infecting dose), the transmission route andthe susceptibility of the respective mass. Each of thesestages in the epidemic chain has its particular featuresfrom country to country. The present study is concer-ned with the analysis of the specific conditions thatfavor the occurrence of the epidemic process, theestimation of the risk degree of transmission of infec-tions caused by hepatitis B, C viruses as well as ofHIV infection in Romania.

As concerning the source of infection, the fol-lowing data are worth mentioning:

- the increasing incidence/prevalence of hepatitisand HIV infections among the adults;

- the increasing number of patients consultingthe dentists;

- the increasing frequency of bleeding proce-dures in Romania caries and their complications area very common occurrence, and periodontal diseasesaffect an important percent of population;

- the numerous visits to the dentist's of HIV-

26

ABSTRACTAs knowledge has accumulated on the blood-transmitted pathogenic agents, the contact with biologicalfluids (blood, plasma, saliva, etc) from apparently healthy individuals has started to be regarded as a realprofessional risk for dentists. Theoretically, exposure to a contaminated biological specimen may have asa consequence transmission of infection from patient to dentist, from dentist to patient and from patientto patient via inadequately decontaminated and sterilized dental equipment. The present study is con-cerned with the analysis of the specific conditions that favor the occurrence of the epidemic process, theestimation of the risk degree of transmission of infections caused by hepatitis B, C viruses as well as ofHIV infection in Romania. The data for the study were collected using two processes. First a self repor-ting survey and secondly an experimental procedure were performed. The testing of dentists' knowledgeof blood transmissible diseases and infection control in their offices were performed using a questionnairewith 129 questions. The professional incidents/accidents representing a potential risk were counted usinga questionnaire (with 37 questions). Serological markers were tested with ELISA kits. The monitoring ofsterilization was accomplished with a questionnaire and biological tests. Many conclusions result fromthe study. There is an extremely reduced probability and infection transmission from the dentist to thepatient. The transmission of infection from the patient to the dentist represents a low risk (for all that, therisk should not be minimized). The rigorous control and observation of infection prevention measures indental offices is necessary to stop the infection transmission from patient to patient. The dentists' post-graduate training in infection control measures should be completed with knowledge regarding the bloodtransmissible infections epidemiology. Learning more about the epidemiological process enables the den-tists to avoid wrong attitudes and behaviors.

CONTROL OF BLOOD-TRANSMITTED INFECTIONS IN DENTISTRY

Eugenia Aurora Neguþ1, Monica Bãlteanu1, G. Ionescu1, A. Bãncescu1, A. Iliescu2, N. Skaug3

1N.I.R.D.M.I. „Cantacuzino“, Bucharest, Romania; 2U.M.Ph. „Carol Davila“, Faculty of Dentistry, Bucharest, Romania;3University of Bergen, Norway

Key words: dentists, risk, blood transmission, prevention, attitude

infected patients: hairy leukoplakia, recurrent ulcera-tions, the rapidly progressing periodontitis, the atopi-cal gingivites, the necrotising stomatites (2,3,4). Atthe same time, consideration should be given also tothe tendency to increase of salivary glands affections,as a direct consequence of infection or of the therapy (5).The treatment of HIV patients' oral affections remains aproblem even in countries with good resources formedical assistance; these patients require a huge vo-lume of medical assistance, not to mention the accep-tability difficulties that are still encountered (6).

As concerning the transmission route:- not all dental cabinets (particularly those in the

rural areas) are adequately equipped (dental devicesand protection equipment);

- the use of the facilities for a correct and moni-tored sterilization may be affected by the high cost ofpower supply.

Regarding the exposed people:- the personal protection equipment (masks, glo-

ves, protection eye glasses) is not always used;- an important percent of the medical staff

refused hepatitis vaccination.Considering these particularities, the present study

is aimed at testing the existent situation in view ofestimating the real cross-infection risk.

2. MATERIAL AND METHOD

The study included 174 dentists from 32 counties,namely 137 from rural area and 37 from urban area.They were randomly selected by the Ministry ofHealth and gave their consent.

The following parameters were included:1. Testing the knowledge of the dentists regard-

ing the blood-transmitted diseases and the control ofinfection in their cabinets. This was achieved by aquestionnaire containing 129 questions regardingthe dentists' personal data, their knowledge on hepa-titis and HIV infections, their attitude toward the pa-tients included in the risk categories, the way inwhich decontamination and sterilization are perfor-med, the use of personal protection equipment, pro-tection through vaccination.

2. Monitoring of professional incidents / acci-dents having a potential risk. During this three-yearstudy, dentists were requested to fill in a new ques-tionnaire for each professional incident / accidentanswering to questions regarding the conditions andcircumstances in which it occurred and the mannerin which it was managed.

3. Testing of the serological markers. The den-tists included in the study were submitted to determi-nations of the serological markers for hepatitis B andC (HBcAb, HBsAb, HBsAg, HBeAg, HBeAb, HCVAb)and HIV infection at the beginning of the study and

three years later. 25 patients of each dentist carrier ofHBsAg or HCVAb were tested for the same serologi-cal markers. From an equal number of dentists withnegative serological markers, the markers of 25 pa-tients were also tested. The tests were performed atthe National Institute of Research-Development for Mi-crobiology and Immunology "Cantacuzino" by ELISA,using the following kits:

ORTHO antibody HBsAg ELISA Test System 3,Hepanostika antiHBc Uni-Form, Monolisa antiHBs3.0, HBeAg/antiHBe MUREX, ORTHO HIV1/HIV2Ab capture.

4. Monitoring of sterilization. Monitoring of ste-rilization equipment (dry heat sterilizers and steamautoclaves) was performed by the control of steriliza-tion using biological indicators (BI) as they are con-sidered to be the closest to the "ideal monitor" for thesterilization process (7). They were used at the firstand the last weekly sterilization, the operation beingrepeated after one month. At each sterilization cycle(a total of four cycles), three BIs were used whichwere placed in boxes or parcels in three differentparts of the sterilizer.

For the dry heat sterilizers with lateral heatingsource, ventilator and several levels, a BI was placedat the lower level, near the sterilizer door, in themedian portion, the second was placed at the back ofthe lower level, opposite the heating source and theventilator and the third was placed at medium level,in the middle of the interior.

For dry heat sterilizers with inferior heating source,with several interior levels, a BI was placed at thelower level towards the rear wall of the equipment, inthe median area, the second one was placed at medi-um level in the middle of the sterilizer, the third wasplaced at upper level, near the door, towards theextremity where the door opened. For the dry heatsterilizers with inferior heating source, equipped withonly one tray, a BI was placed at the back, another inthe middle and the third at the front of the tray.

For steam autoclaves, a BI was placed at the back,another in the middle and the third near the door.

The indicators used were of BAG Bio Strip type;in case of dry heat sterilization, strips impregnated withBacillus subtilis spores were used (2 x 106 spores/strip),and in case of humid heat sterilization (autoclaves)strips impregnated with Bacillus stearothermophilus(1.6 x 106 spores/strip) were used. The strips weresent to NIRDMI "Cantacuzino" were incubated in soyatrypticase broth, 7 days at 30 - 35 oC (Bacillus subtilisspores) and at 55 oC (Bacillus stearothermophilus).

The epidemiology departments within CountyPublic Health Divisions were in charge of distributingthe questionnaires and the BIs as well as sera sam-pling from dentists and patients.

Control of blood-transmitted infections in dentistry

27

The results of the serology tests and of steriliza-tion controls were confidential and transmitted di-rectly to the participants in the study.

3. RESULTS AND DISCUSSION

Of the 500 dentists asked to participate in thestudy, 174 accepted at the beginning of the study.They filled in the questionnaire, were tested for sero-logical markers and indicated the patients who weresimilarly tested. After 3 years, 93 dentists remained inthe study, were serologically retested and participa-ted in the sterilization control and filled in the ques-tionnaires regarding the occupational incidents/acci-dents.

3.1. General data about the participants

Most of them were women (62%), men repre-sented 38% [Q3], 63% were 41 - 60 yrs old, 25%were 31 - 40 yrs old, 12% were 30 - 35 yrs old [Q2],73% were married [Q4]. Most of them had a largeexperience (half of them had over 20 years experi-ence in their profession) [Q5], one third had a spe-cialty (29%0 [Q6]. 70% of the tested dentists worked25 - 40 hours/week, which means 5 - 8 hours/day;most of them (55%) had 10 - 19 patients/week, whichmeans an average of four patients/day, and one thirdhad less than 10 patients/week, which means twopatients/day [Q9]. The dentists that participated in thestudy worked mainly in the rural area (77%) [Q7.1].In most of the cases (86%), one dentist worked in thedental setting [Q8.1.2.]; 93% did not have hygienists[Q8.3] and more than one half did not have a medicalnurse [Q8.3.2.].

3.2. Data about the knowledge on blood-transmittedinfections

When asked about the number of post-universi-ty training hours (in the last two years) regarding thisissue, 74 dentists did not provide any answer, and ofthe 100 who answered, 77 indicated the absence ofthese hours [Q10].

Knowledge about hepatitis BOver 90% of the dentists knew that in at least

50% of cases, those who have hepatitis B do notknow when exposure occurred [Q27], over 70%knew that if contact with blood occurs, the exposedperson must be tested for the presence of antibodiesagainst HBV antigens [Q25] and 43% answered cor-rectly when revaccination must be performed [Q26].All these pointed to the conclusion that their know-ledge regarding HVB infection was "satisfactory".Their knowledge about protection through vaccina-

tion was not just as good. Only 86% answered and ofthese only 16% appreciated correctly that protectionwas in the range of 8 - 15 years. Generally, thoughAbs become undetectable after 12 years, protectionagainst the disease is preserved (8) [Q20].

Knowledge about hepatitis DThe information about the severity of this disease

was insufficient: 60% did not have any notion aboutmortality caused by this infection, 11% considered itless dangerous than hepatitis B and almost one quar-ter thought it resembled hepatitis B [Q31].

Knowledge about hepatitis CThe knowledge about hepatitis C seemed to be

quite insufficient, considering that approx. 75% mis-took the absent or reduced symptomatology of hepa-titis C for that of hepatitis B [Q33], and the evolutionand mortality were correctly appreciated by only60% [Q34], 45% respectively [Q39]. 40% appreciat-ed correctly its contagiousness [Q38] and only 42%indicated all the transmission routes [Q36]. The riskgroups were indicated correctly by only 10% [Q35].Despite this manner of perception of hepatitis Cinfection, an overestimation of the role played bydental treatments in its transmission could benoticed: 90% of the dentists considered that the viruscan be transmitted during these treatments [Q37].

Knowledge about HIV infectionMost of the dentists (92%) appreciated correctly

that neither anamnesis, nor clinical examination canidentify all HIV or hepatitis virus carriers [Q62] andapproximately 82% appreciated correctly the ab-sence of symptomatology for a long period of time inHIV-infected persons [Q63]. The absence of HIVtransmission via saliva was known by only 65% ofthe participants. About 65% indicated correctly thatHIV infection is less infecting than hepatitis B [Q60].The risk of transmission of the disease by injurycaused by a contaminated object was overestimatedand only 10% answered correctly (below 1% con-tamination risk) [Q122].

Although 82% indicated correctly that cross-infection control practices are the same for HIV andhepatitis [Q61] and that dental treatment was appliedby considering all the patients as being potentiallyinfected with these viruses [Q59], 73% consideredthat additional control practices should be used incase of HIV-infected patients, which would be toomuch costly [Q55]. 48% thought that the type of per-sonal protection barriers used depended on the infec-tious status of each HIV- infected patient, and 55%considered that it would depend upon the therapeu-

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NEGUÞ et al.

tic procedure applied [Q57]. Finally, only 58% of thedentists considered that a HIV-infected person couldbe safely treated in their dental setting [Q58].

3.3. The reflection of knowledge about blood-trans-mitted infections in the dental practice

The following were investigated: the attitudetowards hepatitis B vaccination, the attitude towardsthe potential risk represented by risk group patients,the control of cross-infections in the dental practice.

3.3.1. The attitude towards hepatitis B vaccination

Of the dentists participating in the study, 43.7%(76 dentists) were vaccinated and 56.3% (98 dentists)had not been vaccinated [Q12].

For the 98 non-vaccinated dentists, the argu-ments were either real reasons (three of them wereHBsAg carriers and 13 had Ab protective titres), orpersonal conceptions regarding protection (22 den-tists thought that the equipment assures completeprotection and four considered that they knew theirpatients and that none of them could be a source ofinfection). Most of them preferred to minimize theissue and answered: "I had no time" (16 persons), "Imissed from the dental setting" (10 persons), "the vac-cine is not available for me" (8 persons), "I have noresources available" (2 persons), etc. [Q19].

Of the 76 vaccinated dentists, 71% (54 dentists)said that they had been correctly vaccinated, receiv-ing all the three vaccine doses [Q14] by i.m. injectionin the deltoid muscle [Q15]. The results of the sero-logical tests performed during this study indicatedthat only 36 persons were vaccinated (HBsAb presentand HBcAb absent), namely only 47.4% of thosewho had answered in the questionnaire that they hadbeen vaccinated. The explanation for the remaining18 persons in whom HBsAb was not detected couldbe the absence of an immune response ("non-respon-ders") or their vaccination had been performed in1990-1992, and the presence of antibodies becamenow undetectable [Q16]. These suppositions are hardto demonstrate as a serological testing to prove effi-ciency was performed to a very little extent: 8% (6persons) [Q17].

Revaccination was performed to an even lowerpercentage (4%), namely 3 persons [Q18] and thereason was either a long period of time since vacci-nation, or a contamination with blood from a risk per-son [Q19].

As concerning vaccination of the members of theteams in the dental setting, the data are not numer-ous, considering that most dentists worked by them-

selves (86.21%) or with one colleague at the most(11.5%) [Q8.2], that most of them (93%) did not havea hygienist [Q8.3.1.], and 35.6% (61 dentists) hadonly one medical assistant [Q8.3.2.] Of these medicalassistants, 56 (91%) were not vaccinated, excepting 5persons (9%) who had protective antibodies. In mostof the cases (78%), the dentists did not know why themedical assistants had not been vaccinated [Q23],although all the dentists offered to pay the cost oftheir vaccination [Q24].

3.3.2. The attitude towards the potential risk repre-sented by the patients from the risk groups

Asked about the patients they had treated,25.3% of the dentists answered they had treatedpatients with hepatitis B, 11.2% - patients with hepa-titis C, 6% had treated patients with HIV infectionand 15.4% treated patients belonging to a risk group[Q124-127].

About 92% of the dentists answered that it wasimportant for them to know whether the patient wascontaminated or not [Q43], especially HIV-infected[Q45] and 87.8% declared that in anamnesis therewere also questions about hepatitis B and C or aboutHIV infection [Q80]. The percentage of the dentistswho knew nothing about their patients was in therange of 27% - 47% [Q124-127].

The problem is important as the wish to treat riskgroup patients is quite small. The lowest availabilityto treat patients from risk groups (full consent andmoderate consent) was when these patients wereHIV-infected (41%0 [Q71]; followed by those whoused drugs (50%0 [Q65], patients who receiveduntested blood transfusions (53%) [Q67], homo/bise-xuals (57%0 [Q66] and patients with sexually-trans-mitted diseases (58%) [Q68]. The highest availabilitypercentage was reported in hepatitis B patients (46%gave their total consent and only 16% expressed theirdisagreement [Q69] and hepatitis C (44% gave theirtotal consent and only 18% expressed their disagree-ment [Q70]).

If asked about their emotional reaction in case ofa HBV-infected patient 55.4% of the dentists declaredthey had no problem and 44.6% that they had, thesituation is completely different in case of HIV infec-tion. In this latter case, 15% of the dentists refused totreat such patients [Q48], 19% thought they had noethical responsibility to treat this category of patients[Q49] and preferred to send them to be treated byother colleagues (21%) [Q47] or consider that specialclinics should be set up for the treatment of those withno signs of the disease (54%) [Q53] and particularly ofthose with a generally affected medical condition (90%)[Q54]. This comes in contradiction with the affirmation


29

of 58% of the dentists who said that they could safelytreat an HIV+ patient in their dental setting.

The reason why the availability to treat HIV-infected patients is quite low can be explained by:

- 62% think that if they treated HIV-infected orAIDS patients, they would be placed in the group ofthose who are at high risk of getting the disease[Q50]. When becoming a patient, the dentist himselfis reluctant to be treated by a dentist who has HBV,HCV or HIV-infected patients; only 45% of the den-tists would go to such a dental setting [Q72] and if thepatients of such a setting would be HIV+ only 39%[Q73]. On the other hand, only 63% of the dentiststhink that it is the right of patients to know the healthcondition (infected/non-infected) of the dentist; 15%think that it is unnecessary and 12% are not surewhether this is necessary [Q46].

- 43% think that it would be difficult for them tocollaborate with the members of the team because oftheir fears [Q51]. The percentage is obviously exa-ggerated, considering that in 86% of cases there isonly one dentist in the dental setting [Q8.1.2.] andmore than half of the dentists do not have a medicalassistant [Q8.3.2.].

- 35% think that the patients would refuse tocome to their practice [Q52].

However, 85% of the dentists declare that theywould request the serological testing of the patient incase of a contamination risk exposure [Q44].

3.3.3. The control of cross-infections in the dentalpractice

To have a complete image of the control ofcross-infections, several methods were used:

- filling in questionnaires regarding aspects of themanner in which the dentist controls the epidemio-logical process (general rules, personal protectionequipment, behavior, decontamination) or data refer-ring to the eventual accidents/incidents occurringduring the three years of the study;

- monitoring sterilization of the equipment using BIs.

3.3.3.1. Data regarding the extent to which the ge-neral rules of infection control are observed

The answers provided point to the fact that 67%of the dentists have knowledge of the universal pre-cautions [Q78], and over half of them (60%) knowthe regulations imposed at national level regardinginfection control [Q75]; 27% have a manual aboutinfection control [Q76], and 91% of the medicalassistants comply with its requirements [Q77].

Although previously 82% [Q59 and Q 60] hadthought that all the patients must be practically con-sidered as potentially infected and that the infection

control practices are the same for any patient, in prac-tice they declare that they use special measures incase of HIV-infected (45%) [Q81] or HBV infected(50%) [Q82] , though 73% had thought that thesespecial measure are extremely expensive [Q55].

3.3.3.2. Data regarding the self protection and theprotection of the auxiliary personnel

89% of the dentists wear a gown [Q113], 64%wear a mask [Q110], 62% wear protection eye glass-es [Q112], and 48% wear gloves [Q99]; 16% of thedentists declared they had an allergy to latex [Q95],although they were not submitted to special tests.

Only 24.5% of the dentists use an aseptic solu-tion for mouth rinsing before any intraoral procedure[Q105]. The saliva aspirator is always or most of thetime used by 43% of the dentists [Q115] and the highvolume aspirator (surgical) was used by only 7.5% ofthe dentist [Q114]; 72% of the dentists never use diga[Q116]. The dental prints and the prosthetics arealways or most of the time decontaminated by onlyhalf of the dentists (52%) before being sent to the tech-nician [Q117].; prosthetics removed from the oral cav-ity during adaptation are always or most of the timedecontaminated by 68% of the dentists [Q119].

Dentists always or usually have (85.3%) a spe-cial container for the sharp disposable equipment[Q106]; but 36% put the cap on the needle for anes-thesia directly with the hand [Q108]; 40% declarethat they always or usually do this procedure with ahaemostatic or a special device [Q109].

3.3.3.3. Data regarding patient protection

All the dentists used to wash their hands beforetreating each patient [Q98], but only 97% did it beforeusing gloves [Q100]. 78.3% of the dentists always orusually changed gloves after each patient and only38% changed the mask after each patient [Q111].

83% of dentists always or usually left the waterin the dental unit run for a few seconds [Q102].

77.6% of the dentists disinfected the dentalprints and the prosthesis after receiving them from thetechnician [Q118], and 80% of the dentists decon-taminated them before placing them in the oral cavi-ty [Q120].

The disposable equipment was used in only41.4% of the dental settings [Q104] and only 9% ofthe dentists sterilized the counter angle and otherdevices after each patient [Q103].

Because of the insufficient number of answers,no conclusion can be drawn about the dentists whoused boiling of the dental instruments or who pre-pared correctly the materials for sterilization (previ-ous washing) [Q91].

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As concerning sterilization, 166 dentists (95%)used dry heat [Q83], 22 (14%) used humid heat[Q86], and 29 (29%) used other types of sterilization(u.v., formalin, quartz).

The answers showed that of the 166 dry heatovens less than a half 72 (43.4%) were submitted tocontrol; most of these 59 (82%) were controlled usingchemical indicators [Q84], and only 13 (18%) usingBI [Q85]. Of the 59 dry heat ovens controlled usingchemical indicators, only 25 (42%) were daily con-trolled, and of the 13 controlled by BI only 2 (15.4%)were daily controlled.

Of the 22 autoclaves, 15 (68%) were controlledfor the efficiency of sterilization [Q86]: 11 (73%)were controlled using chemical indicators [Q87] and4 (27%) with BI [Q88]. Of the 11 autoclaves chemi-cally controlled, three were daily controlled, and ofthe 4 autoclaves submitted to biological control, twowere weekly controlled. In the remaining cases, thecontrol is performed annually (Table 1a).

The analysis of the given answers showed thatsterilization was not adequately monitored. Even whenit was performed, chemical indicators (which signalonly the reaching of a certain temperature) were pre-ponderantly used, but not at each sterilization cycle.

Only 91 dentists who used dry heat ovens(54.82%) and 6 dentists that used autoclaves (27.27%)agreed to participate in the control performed duringthe study. In case of dry heat ovens, three of the den-tists did not observe the control schedule, namelythey did not use 3 BI strips at each sterilization; undersuch conditions, only 88 dry heat ovens and 6 steamautoclaves were included in the study; four steriliza-tion cycles were checked for each of them.

The results were as follows:

Dry heat sterilizers- in ten of the sterilizers, all the four sterilizations

were correctly performed (11.4%);- in 78 (88.6%) of the sterilizers, such sterilization

deficiencies were detected:- 16 (20.51%) sterilizers had a defective sterili-

zation;- 15 (19.23%) devices had two defective steri-

lizations- 16 (20.51%) devices had three defective steri-

lizations- 31 (39.74%) devices had four defective steri-

lizations

Steam autoclavesAs concerning the autoclaves, six dentists

observed the control schedule. In two cases, all thesterilizations were correctly performed, but in fourcases the 120 degrees C temperature was not reachedfor the necessary duration (Table 1b).

The analysis of the results obtained leads to theconclusion that there are no significant differences inthe manner in which sterilizations are performed at thebeginning or at the end of the week: thus, for dry heatovens, 66 (37.9%) correctly performed sterilizationswere reported at the beginning of the week and 67(38.5%) at the end of the week; for autoclaves, similarfigures were reported, namely five correct sterilizationsboth at the beginning and at the end of the week.

Generally, this means that out of 188 sterilizers,94 (50%) were controlled and of these only 12 (12.8%)performed a correct sterilization, as indicated by thefour controls. This points to severe deficiencies in the


31

Table 1a - Control of sterility reported by the dentists

Table 1b - Control carried out during the study using biological indicators

control of cross-infections. Deficiencies are related toboth the low level of knowledge regarding the correctsterilization and the transmission of the various typesof infections as well as to the wish to increase the effi-ciency of the dental setting by reducing costs.

3.3.3.4. Data on occupational accidents

For a better understanding of the causes of occu-pational accidents, dentists received a special ques-tionnaire with questions referring to the accidentsthat occurred during the study. Unfortunately, it seemsthat the duration of the study was too long, thus, at theend of the three-year study of 174 participants, only 93accepted to answer the questions. Of these, 37(approx. 40%) reported occupational accidents.

Most of the dentists that reported occupationalaccidents (57%) had 20 -30 years of experience [Q5],worked 25- 40 hours per week (69%) and declared(73%) they had had previous exposures [Q7]. Most ofthe accidents (73%) occurred at the beginning and atthe end of the week [Q8], between 10.00 - 13.00 hrs(70%) [Q4].

The main activities being performed during theoccurrence of the accidents were treating and con-sulting the patient in the absence of an assistant(42%) or in his presence (25%) [Q10].

The therapeutic acts during which accidentsoccurred most frequently were the endodontic pro-cedures (16%) and the cleaning of the dental instru-ments after treatment (16%), tooth extractions (15%)and the cleaning of the dental instruments duringtreatment (11%) [Q20].

The accident occurred most frequently bysplashing intact skin with saliva (27%), or stingingwith the drill (19%), with the syringe needle (16%) orwith an endodontic instrument (13%) [Q15].

Exposures to saliva (33%), blood (31%) or con-taminated dental instruments/prosthesis (27%) [Q11]were reported and the most exposed regions were thefingers (55%), the left hand (21%) or the right hand(15%) [Q14].

Most of the dentists appreciated that the woundswere superficial, accompanied (64%) or not (32%) bybleeding [Q16], and the intensity of contamination,judged by the amount of blood/saliva with whichcontamination produced was in 79% of cases verylow [Q17].

The protection equipment worn at the momentof the accident was the gown (30%), the eye glasses(25%), the mask and the surgical gloves (20%) [Q21].Of those who had an occupational accident, 87%thought they were responsible for it [Q13]. Most ofthe dentists considered that the accident occurredbecause of the difficulty to approach the region where

the therapeutic procedure was performed (41%), fol-lowed by distracted attention (24%), haste or stressdue to the therapeutic procedure (9%) [Q22].

After the accident, dentists take not only imme-diate measures but also measures aimed at preven-ting the occurrence of a new accident. Immediatemeasures consisted in cleaning the area (wound), dis-infecting it and, if needed, dressing it. Treatment afterexposure was confirmed by 38% of the injured den-tists [Q19], 72% (27 dentists) indicated previous de-contamination of the exposed site [Q18], with a dura-tion of maximum 5 minutes between decontamina-tion and exposure for 57% [Q18.1A]. Water and soapwere the decontaminating agents most frequentlyused (41% of cases), followed by alcohol (23%) oriodophor (21%0 [Q18.B]. Among of the immediatemeasures 87% of the responders confirmed no longeruse of the dental instruments implicated in the acci-dent [Q22.II] . Avoidance of a new accident wasrelated to a better concentration (27%), the use ofmore efficient protection equipment (21%) as well asmeasures aimed at preventing stress (19%) [Q23].

According to the answers given, only 17 (46%)of the 37 dentists who had accidents were vaccinatedagainst hepatitis B, one dentist had an incompletevaccination and 3 (8%) had hepatitis B in history[Q24]. Of the 17 vaccinated dentists, only 6 (35,3%)were serologically tested for the efficiency of vacci-nation [Q25], although this test should have beenperformed 2 months after vaccination (8). Over onethird of the injured dentists had had a tetanus revac-cination in the last 10 years [Q26].

The information about the risk represented bythe condition of the patient during whose treatmentthe accident occurred was unsatisfactory. 34- 40% ofthe dentists had such information; they did not knowwhether the patient was haemodialysed (28%) [Q34],if he came from an endemic region for hepatitis B(31%) [Q33], if he was HIV-infected (42%) [Q30] orAIDS -confirmed (39%) [Q29]. Of the injured den-tists, 50% did not know if the patient who might havecontaminated him had shared syringes with otherpersons [Q32], 58% did not know whether the pa-tient was HBV-infected [Q27], HCV-infected [Q28] orif he had received any transfusion [Q31]. The dataprovided by the injured dentists confirmed the dataprovided by the study of the 174 dentists initiallyenrolled in the study: the percentage of those whohad no information about their patients varied from27% to 47%; the dentists who knew that they pro-vided treatment to patients belonging to risk groupswere in the range of 6 - 25.3%. Although 85% hadinitially declared that they would request testing ofthe patient in case of an accident, none of them madesuch a request.

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This explains why the measures taken in case ofan accident referred mainly to first aid procedures; ifa serological test was requested, it involved especial-ly the dentist. Hepatitis B revaccination, administra-tion of zidovudine were very rarely mentioned [Q128].Though occupational accidents were reported, theguide indicating the measures in case of an accident(injury) missed in 81% of the dental health care set-tings [Q121], and the accidents were not reported in62% of cases [Q129].

3.4. Control of cross-infections by the study of theserological markers for hepatitis and HIV infections inthe dentists included in the study and in their patients

With their consent, from 174 dentists blood sam-ples were take and the serological markers were deter-

mined for HBV, HCV and HIV infections. Th resultsare presented in Table 2 and Table 3. The presence ofhepatitis B in history was a common occurrence43.1%. In the USA, before the introduction of vacci-nation, prevalence of hepatitis B was of 14%, aftervaccination and universal precautions it dropped to9% (9). Prevalence of hepatitis C among the dentistsin Romania was double as compared to the USA (4%versus 1-2%) (10,11). No cases of HIV infection wasreported among the dentists enrolled in the study.

The risk of HBV or HCV infection transmissionfrom dentist to patient

As the HBsAg carriers were very few among thedentists enrolled in the study (3.4%) and the HBeAgcarriers were not detected, the risk that these dentistsmight be an infection source is very unlikely (Table 2and Table 3).


33

Table 2 - Results of the serological tests in 174 dentists

Table 3 - Situation of dentists with hepatitis B infection

Table 4 - Distribution of hepatitis B and C infection patients depending upon the dentist

It is known that the risk to develop clinical hep-atitis B following contamination with HbsAg andHbeAg positive blood-stained instruments proved tobe 22 - 31%, whereas contamination with HbsAgpositive blood only was of 1 - 6% (12).

The risk of transmission from dentist to patientwas studied also by the analysis of serological mark-ers of the patients of dentists carriers of HbsAg orHCVAb as compared to those of the patients of den-tists with no hepatitis infections (Table 4).

20-22 patients were studied from each dentistcarrier of HBsAg or HCVAb as well as for the samenumber of non-carrier dentists; this led to the testingof a total number of 563 patients. No increased inci-dence of HBsAg carriers was noticed among thepatients of the carrier dentists of this antigen; this inci-dence was of 26.8% against 38.5% in the patients ofthe dentists carriers of HCVAb or 36.9% in the pa-tients of the dentists with no hepatitis infections. Si-milarly, the patients of the carrier dentists of HCVAbhad a prevalence of the HCV infection of 3.84% ver-sus 7.21% in the patients of the dentists carriers ofHBsAg or 3.3% in the patients of the non hepatitis

infected dentists. There were no cases where patientswith HBsAg or HCVAb belonged to a single dentistcarrier of HBsAg or HCVAb, respectively. The dataconfirmed the results obtained in other studies re-garding the extremely low level of transmission of theinfection from the infected dentist to a patient (12).

Risk of infection transmission from patients todentists

This risk could be appreciated according to theresults presented in Table 5.

The comparison of the percentage of positiveserological markers in the group of patients and thegroup of dentists revealed similar values for the HbsAg,HbeAg and HCVAb carriers, which did not indicatethe possibility of frequent transmission. Table 6 clearlyshowed that during the three-year study, no modifica-tion of hepatitis B or C incidence was reported in the93 dentists that remained in the study.

The data confirmed the studies of other authorsregarding the transmission of infection from patient todentist (1). The studies carried out at internationallevel revealed that HCV is not efficiently transmittedduring professional exposures to blood (13, 14, 15,

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NEGUÞ et al.

Table 5 - Presence of serological markers for hepatitis B or C and HIV infection in the group of dentists and the group of their patients

Table 6 - Comparative presence of serological markers at the initial testing and three years later (group of 93 dentists)

16), the prevalence of HCV infection in dentists, sur-geons and other hospital care workers being identicalwith that in the general population (10, 11).

Although the problem of infection transmission isnot negligible if consideration is given to several data:

- The extremely low percentage of vaccinatedpersons (9.76%) among patients (Table 5);

- The high percentage (7.28%) of patients in theimmunological window stage, which points to anactive increase of hepatitis B infection incidence inpopulation (Table 5);

- The high frequency of occupational accidentsidentified during the three-year study (40% of thedentists had accidents, of which 64% involvingbleeding), the accidents that occurred due to the mis-use of the protection equipment and of the deficientpost-exposure management (17) suggest the presenceof conditions that favored the transmission of infection.

HIV infection testsAs concerning HIV infection, although there were

no infection cases among the dentists or the patients,the risk remains as the infection prevalence increases.Even though estimated to be very low in other coun-tries (18), 0.3% after percutaneous exposure (19) and0.1% after mucous membrane splashing (20), in theexisting conditions all the stipulated precautions mustbe strictly taken. The use of the protection equipmentis necessary. There were cases of blood splashing ofconjunctiva (21) or of concomitant transmission ofhepatitis C and HIV after exposure of the skin (22).

Hepatitis B vaccinationThe percentage of persons vaccinated for hepati-

tis B in the group of dentists was not very high: 20.7%.The tests performed after three years showed thatonly 8.6% of the dentists presented detectable anti-body titre (Table 6). Although protection remainspresent for another 2-3 years, the need for revaccina-tion becomes evident, as well as that of testing theefficiency of vaccination (8).

4. CONCLUSIONS

1. The study proves the extremely low possibili-ty to transmit the infection from dentist to patient byhis contact with blood from an infected dentist. Thisis well demonstrated by the very low percentage ofHbsAg carriers (4.3 %) and the absence of HbeAg car-riers. On the other hand, the percentage of patientswith hepatitis B or C infection per dentist does notpresent any correlation with the respective dentist'shealth state (HbsAg or HCVAb carrier or with no hep-

atitis infection).2. The risk of transmission of an infection from

patient to dentist is also low: the small number ofHbsAg carriers (3.5%) and the even lower number ofHbeAg carriers (0.5%), the unchanged incidence ofhepatitis infections after three years in the dentistsgroup, the accidents reported in this period whichdid not meet in any of the cases the conditions oftransmission. But this should not lead us to think thatthe risk can be minimized because:

- The very low protection of population: the per-centage of vaccinated persons being very low (9.76%)

- The "active" increase of hepatitis B in population(7.5% were found in the immunological "window")

- The high frequency of occupational accidentsinvolving bleeding

3. A strict control of the observance of preven-tion measures of infections in dental health care set-tings aimed at blocking the transmission of infectionsfrom patient to patient via dental instruments. A cor-rect monitoring of sterilization should be introduced;every time it is possible, besides the chemical indica-tors, biological indicators will be used to appreciatethe efficiency of the sterilization process. The dentalhealth care settings must have documents regardingthe rules for the control of infection in dental settings.

4. Post-university training of dentists will becompleted with more strict notions of the epidemio-logy of blood-transmitted infections. Thus, the under-estimation of the risk (the lack of anamnesis regarding"risk states") as well as the overestimation of the risk(the transmission of hepatitis C and HIV infection)will be avoided. The correct understanding of the si-tuation will lead to a correct attitude regarding thepossibility to treat patients included in the "risk" cat-egories, to the adoption of all the control measuresand last but not least the modification of the concep-tion regarding hepatitis B vaccination (the need for acorrect vaccination, checking its efficiency and timingof revaccination).

REFERENCES

1. Centers for Disease Control and Prevention.Control in dental health-care settings. MMWR2003;52 (RR-11).

2. Winkler JR, Murray PA. Periodontal disease: apotential intraoral expression of AIDS may be arapidly progressive periodontitis. California DentalAssociation Journal 1987;15:20-4.


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3. Williams CA, Winkler JR, Grassi M, Murray PA.HIV associated periodontitis complicated by ne-crotising stomatitis. Oral Surgery, Oral Medicineand Oral Pathology 1990;69:351-355.

4. Winkler JR, Robertson PB. Periodontal diseaseassociated with HIV infection. Oral Surgery, OralMedicine and Oral Pathology 1992;73:145-150.

5. Hodgson TA, Greenspan D, Greenspan JS. Orallesions of HIV disease and HAART in industializedcountries. Adv Dent Res 2006;19:57-62.

6. Robinson PG. Implications of HIV disease for oralhealth services. Adv Dent Res 2006;19:73-79.

7. Widmer AF, Frei R. Decontamination, Disinfec-tion and Sterilization. In: Murray PR, editor. Ma-nual of Clinical Microbiology. 7th ed. WashingtonDC: ASM Press, 1999:138-164.

8. Centers for Disease Control and Prevention. Im-munization of health care workers: recommanda-tions of the Advisory Committee on ImmunizationPractices (ACIP) and the Hospital Infection ControlPractices Advisory Committee (HICPAC). MMWR1997;46(RR-18).

9. Cleveland JL, Sien C, Lockwood SA, Gruninger SE,Goolm BF, Shapiro CN. Hepatitis B vaccinationand infection among U.S. dentists 1983-1992. JAm Dent Assoc 1996;127:1385-90.

10. Thomas DL, Gruninger SE, Siew C, Joy ED, QuinnTC. Occupational risk of hepatitis C infectionsamong general dentists and oral surgeons in NorthAmerica. Am J Med 1996;100:41-5.

11. Lodi G, Bez C, Porter SR, Scully C, Epstein JB.Infectious hepatitis C, hepatitis G, and TT virus:review and implications for dentists. Spec CareDentist 2002,22(2):53-58.

12. Werner BG, Grady GT. Accidental hepatitis B sur-face antigen positive inoculations: use of e antigento estimate infectivity. Am Intern Med 1982;97:367-9.

13. Alter MJ. The epidemiology of acute and chron-ic hepatitis C. Clin Liver Dis 1997;1:559-68.

14. Puro V, Petrosillo N, Ippolito G. Risc of hepati-tits C seroconversion after occupational exposuresin health care workers: The Italian Study Group onOccupational Risk of HIV and Other BloodbornInfections. Am J Infect Control 1995;23:273-7.

15. Lanphear BP, Linnemann CC, Canon CG,DeRonde MM, Pendy L, Kereley LM. Hepatitis Cvirus infection in health care workers: risk of expo-sure and infection. Infect Control Hosp Epidemiol1994;15:745-50.

16. Mitsui T, Iwano K, Masukok et al. Hepatitis Cvirus infection in medical personal after needle-stick accident. Hepatology 1992;16:1109-14.

17. Centers for Disease Control and Prevention.Updated U.S. Public Health Service guidelines forthe management of occupational exposers to HBV,HCV and HIV and recommendations for postexpo-sure prophylaxis. MMWR 2001;50(RR-11).

18. Do AN, Ciesielski CA, Metler RP, Hammett TA,Li J, Fleming PL. Occupationally acquired HIVinfection: national case surveillance data during20 years of the HIV epidemic in the U.S. InfectControl Hosp Epidemiol 2003;24:86-96.

19. Bell DM. Occupational risk of HIV infection inhealth care workers: an overview. Am J Med1997;102(5B):9-15.

20. Ippolito G, Puro V, De Carli G. The risk of occu-pational HIV in health care workers: ItalianMulticenter Study, The Italian Study Group onOccupational Risk of HIV Infection. Arch InternMed 1993;153:1451-8.

21. Sartori M, La Terra G, Aglietta M, Manzin A,Navino C, Verzetti G. Transmission of hepatitis Cvia blood splash into conjunction. Scand J InfectDis 1993;25:270-1.

22. Beltrami EM, Kozak A, Williams IT et al. Trans-mission of HIV and hepatitis C virus from a nursinghome patient to a health care worker. Am J InfectControl 2003;31:168-75.

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INTRODUCTION

A large variety of viruses (including the influenzaviruses, RSV, parainfluenza viruses, adenovirus, rhi-noviruses, and coronaviruses) cause the clinical syn-dromes that can be associated to diseases of the upperrespiratory tract (rhinitis, nasopharyngitis, laryngitis)or with the ones of the lower respiratory tract (tra-cheitis, bronchitis, pneumonias). However, approxi-mately 50% of the pneumonia cases acquired in anadult community and 15%-35% of the bronchiolitisand pneumonia cases in children are outside a defi-ned etiology.

hMPV was first isolated in 2001 from a nasopha-ryngeal aspirate from an infant in the Netherlands (1).As of the first reporting in 2001, the hMPV was dis-covered in North America (2), Europe (UK, France,Italy, Germany, Spain, Finland) (3, 4), Asia (Hong Kong,Japan) (5), Australia (6). The virus was identified innon-immuno-compromised children, as well as inchildren infected with AIDS in South Africa.

The epidemiological investigations indicatedthat the hMPV infections have a seasonal distribution,overlapping the RSV circulation, most cases beingreported during the winter and early spring months,sometimes extended during the late spring, when theepidemic season of the RSV infections is concluded.

From the epidemiological point of view, the hMPViruses are divided into two subgroups (A and B),

respectively, into four subtypes (A1, A2, B1, B2), thetwo groups circulating simultaneously (6).

The hMPV genome consists of an (-) sense single-stranded RNA of approximately 13 kb, containing co-difying genes for the nucleoprotein (N), the phos-phor-protein (P), the matrix protein (M), the fusionprotein (F), the transcription elongation factor, theRNA synthesis adjustment factor (M2), small surfacehydrophobic proteins (SH), the major attachment gly-coprotein (G) and the polymerase major subunit (L),arranged in the order 3'-N-P-M-F-M2-SH-G-L-5' simi-lar to APV.

There are two major differences between thehMPV genome and the RSV genome: the gene orderis different, and the hMPV genome does not containnon-structural genes

The objective of the present study was to establi-shed whether hMPV is present in Romania and iscausing disease to children.


1. Patients and samplesPatients - in our study, 28 patients were investi-

gated having the respiratory virosis diagnosis. The ageof the patients was between 9 months and 6 years.

Samples - pharyngeal exudates introduced intransport medium till laboratory where they are dis-charged in the medium and frozen at - 80 oC till the

37

ABSTRACTThe human metapneumovirus (hMPV) was first isolated in 2001 in the Netherlands (Van der Hoogen and col-laborators) from a nasopharyngeal aspirate sampled from an infant. Based on the morphological, biochemicaland genetic characteristics, the hMPV was initially classified in the genus Metapneumovirus with the avianmetapneumovirus (APV), the agent causing the respiratory infections of the upper tract in turkeys and otherbirds. Subsequently, together with the respiratory syncytial virus (RSV), it was classified in the Pneumovirusgenus which is a part of the Pneumovirinae subfamily, the Paramyxoviridae family. The aim of the present study was to optimize hMPV molecular detection and to detect the virus in samplesform children with respiratory infections in Romania. Two types of RTPCR commercial kits were evaluated forthe detection of hMPV. Tests were performed on 28 pharyngeal exudates from children aged from 9 months to 6 years, which werenegative for influenza viruses and for Respiratory Syncytial Virus (RSV). Among the tested samples 7 (25%)have been positive for hMPV by RT-PCR. These results document for the first time that hMPV is circulating inRomania and causes respiratory infections, especially in newborns and children under 6 years old.

FIRST DETECTION OF HUMAN METAPNEUMOVIRUS IN CHILDREN WITH RESPIRATORY INFECTIONS IN ROMANIA

Cristina Þecu1, D. Orãºanu2, Aurora Sima2, Maria Elena Mihai1, V. Alexandrescu1, D. Matei3 and Narcisa Samoilã1

1N.I.R.D.M.I. „Cantacuzino“; 2„Grigore Alexandrescu“ Children's Hospital; 3„Alfred Rusescu“ Mother and Child Protection Institute

Key words: a

extractions for RT-PCR (Reverse Transcription Poly-merase Chain Reaction) were made.

2. METHODS

There are no tests available for the antigen fastdetection and due to the difficult growth on cellularsubstrate (LLC-MK2 = tertiary monkey kidney, withthe occurrence of a non-specific cytopathic effect 17

days as of the inoculation), and the diagnosis by RT-PCR represents the most widely available method,with a high sensitivity and specificity. The hMPV po-sitive control was the RNA extracted from an hMPVstrain cultivated on LLC-MK2 cells and was receivedfrom the CHRU Molecular Virology Laboratory -Caen France.

The extractions of the samples was made withQIAamp Viral RNA Mini Kit (cat no 52906) from

38

TECU et al.

Fig. 1 - Target region for RT-PCR

Fig. 2 - The sensitivity of the detection of hMPV with Promega Kit, 2006Legend: Neg. C.= negative control, Pos. C. = Positive control, dilutions of positive control: 1/10, 1/100, 1/1000, 1/10.000, 1/100.000, C.Kit = Kit control from Promega kit

Fig. 3 - The sensitivity of the detection of hMPV with Qiagen KitLegend: undil. = undiluted positive control, dilutions of positive control 1/10, 1/50, 1/100, 1/150, 1/200, 1/250, 1/500, 1/103, 1/104 , neg.c. = negative control

QIAGEN. The primers used for the hMPV detection(which amplified the M gene - Fig. 1) were:

- M1: 5' CCC TTT GTT TCA GGC CAA C 3'- M2: 5' GCA GCT TCA ACA GTA GCT G 3'

The target region for the molecular detection isrepresented in Fig 1. (1)

The mix solution for the reaction (for 1 tube of25 µl) for the Promega kit: water DNase-free 12.6 µl;

First Detection of Human Metapneumovirus in Children with Respiratory Infections in Romania

39

Table 1 - Data on the hMPV positive patients

Fig. 4 (a and b) - Electrophoresis of amplification products from RNA extracted from nasopharyngeal exudates of patientsLegend: 51- 78 samples from children with respiratory virosis, 53+; 56+; 58+; 62+; 64+; 71+; 75+ = positive samplesNC = negative control, PC = positive control

(a)

(b)

buffer PCR 5x 5 µl; dNTP (10 mM) 0.5 µl; MgSO4(25 mM) 1 µl; AMV RT (5U/µl) 0.5 µl; Tfl DNA Pol(5 U/µl) 0.5 µl; primer M1 (10 µM) 1.2 µl; primer M2(10 µM) 1.2 µl.

The mix solution for the reaction (for 1 tube of25 µl) for the Qiagen kit: water DNase-free10.1 µl;buffer PCR 5x 5 µl; dNTP (10 mM) 1 µl; Q solution5x 3 µl; Mix Enzymes 0.5 µl; primer M1 (10 µM)1.2 µl; primer M2 (10 µM) 1.2 µl.

To the 22.5 µl of mix 2.5 µl of RNA extractedfrom the samples of the patients suspected of hMPVinfection are added.

The reverse transcription and amplification pro-gram used with the Promega kit was: 30' - 50 oC; 2' -94 oC; 40X (30" - 94 oC, 30" - 55 oC, 1' - 68 oC); 10'- 68 oC; - 4 oC.

The reverse transcription and amplification pro-gram used with the Qiagen kit was: 30' - 50 oC; 15'- 94 oC; 40X (30" - 94 oC, 30" - 55 oC, 1' - 72 oC); 10'- 72 oC; - 4 oC.

The visualisation was made on a 1.5% agar gelin TBE 1x u ethidium bromide; the migration was per-formed slowly at 150V for approximately 45 minutes.

A bande of 460 pb is a positive result for infec-tion with hMPV.

RESULTS

The sensitivity of this method was tested, in thelaboratory, in parallel with 2 commercial kits (Fig. 2and Fig. 3).

The reaction is more sensitive when workingwith the Qiagen kit, the positive control was detectedfor the 10-4 dilution compared to 10-2 when workingwith the Promega kit.

The amplification with the Qiagen kit was cho-sen to test samples from patients. Out of the 28 testedsamples 6 were positive for hMPV (21%) and a sam-ple was positive both for hMPV, as well as for VRS(Fig. 4,5).

The protocol using the Promega kit did not de-tect hMPV in any of the patients samples.

Data on the hMPV positive patients are presentdin Tabel 1.

CONCLUSION

These results reveal that hMPV is circulating inRomania and is causing respiratory infections in chil-dren.

REFERENCES

1. Van den Hoogen BG, De Jong JC, Groen J et al. -"A newly discovered human pneumovirus isolat-ed from young children with respiratory tract dis-ease", Nat. Med. 2001, 7, 719-24

2. Ann R. Falsey, Dean Erdman, Larry J. Andersonand Edward E. Walsh - "Human Metapneumo-virus Infections in Young and Elderly Adults", TheJournal of Infectious Diseases 2006; 187: 785-790

3. Maria Rosa Lopez-Huertas, Inmaculada Casas,Belsy Acosta-Herrera, Maria Luz Garcia, MariaTeresa Coiras, Pilar Perez-Brena - "Two RT-PCRbased assays to detect human metapneumovirusin nasopharyngeal aspirates". Journal of Virolo-gical Methods, 2005, 129, 1-7

4. Joanne Stockton, Yain Stephenson, Douglas Fle-ming and Maria Zambon - "Human Metapneumo-virus as a Cause of Community - Acquired Res-piratory Illnes", Emerg. Infect. Dis., 2002 Sept.,Vol. 9, No. 9

5. Paul K.S. Chan, John S. Tam, Ching-Wan Lam,Edward Chan, Alan Wum Chi-Kong Li et al. -"Human Metapneumovirus Detection in Patienswith Severe Acute Respiratory Syndrome". Emerg.Infect. Dis., 2003 Sept., Vol. 9, No. 9

6. Theo P. Sloots, Ian M. Mackay, Seweryn Biala-siewicz, Kevin C. Jacob, Emily Mc. Queen et al. -"Human Metapneumovirus, Australia, 2001 -2004". Emerg. Infect. Dis., 2006 August, Vol. 12,No. 8.

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88

INTRODUCTION

Urinary tract infection (UTI) is among the mostcommonly diagnosed bacterial infections of childhood(1). Although frequently encountered and well resear-ched, diagnosis and management of UTI continue tobe a controversial issue with many challenges for theclinician. In infants and young children, UTIs may beharder to detect because symptoms are less specific. Infact, fever is sometimes the only symptom and nuclearrenal scans suggest that the vast majority of febrileyoung children with UTI have pyelonephritis, puttingthem at risk for renal scarring and the long-term seque-lae of hypertension and renal failure. Thus, it is impe-rative that physicians identify these children to instituteearly treatment, evaluate the urinary tract, and monitorfor recurrent UTI.

E. coli (2) causes about 80% of UTIs. Uropatho-genic strains of E. coli are believed to display a varietyof virulence properties that assist in colonization ofhost mucosal surfaces and in circumventing host de-fenses to allow invasion of the normally sterile urinarytract. A limited number of virulence factors, includingadhesins, siderophores, toxins, capsules, and a pro-tease, have been implicated as important traits allowinguropathogenic E. coli to cause disease (3). In several

cases, pathogenicity has been correlated with the pres-ence of genes encoding virulence factors organized onlarge blocks, called pathogenicity islands (PAIs) (4).

The present study was undertaken to evaluate theintrinsic virulence potential of E. coli uroisolates obtai-ned from, taking into account that the severity of theUTI depends both on the virulence of the infectingbacteria and on the susceptibility of the host. To ourknowledge, this is the first molecular study of E. coliurinary isolates from Romanian pediatric cases of UTI.


Patients and Bacterial strainsThe E. coli strains were isolated from 54 children

with ages between 3 weeks and 17 years hospitalizedin the Pediatric Emergency Hospital "Sf. Ioan" fromGalati. Children's records included UTI signs andsymptoms (e.g. dysuria, frequency, dribbling/hesitan-cy, enuresis after successful toilet training, malodorousurine, hematuria, abdominal/suprapubic pain), as wellas systemic signs and symptoms (e.g. fever, vomiting /diarrhea, flank/back pain).

Midstream clean-catch specimens were analysedfrom the children with urinary control, while sterilebag specimens were accepted for the children with no

41

ABSTRACTThe urinary tract is among the most common sites of bacterial infection and E. coli is by far the most com-mon infecting agent in children and adults of both sexes. In an attempt to evaluate the intrinsic virulenceof E. coli uroisolates from children, 54 strains were assessed by using PCR for the presence of five repre-sentative genetic determinants coding for adherence systems (pap, sfa/foc, afa), and toxins (hly and cnf).The prevalence of pap, sfa/foc and afa genes was 55%, 54%, and 44%, respectively. Hemolysin-encodinggene hly was detected in 55% strains, while cnf was exhibited by 35% of the screened E. coli isolates.Among the 39 PCR positive strains isolated from children's urine cultures the co-occurrence of the varioustargeted virulence genes was detected in 30 strains, the virulence profiles identified suggesting the pre-sence of their localization on chromosomal regions known as pathogencity-associated islands. The rapidand reliable detection of the intrinsic virulence potential by this molecular approach could be very usefulwhen evaluating the importance of microorganism pathogenicity versus host's susceptibility for developingan overt symptomatology of infection.

VIRULENCE CHARACTERISTICS OF ESCHERICHIA COLI ISOLATES FROM CHILDREN WITH URINARY TRACT INFECTIONS

Caliopsia Florea1,2, Codruþa-Romaniþa Usein3, Maria Condei3 and Maria Damian3

1Faculty of Medicine and Pharmacy Galati; 2Pediatric Emergency Hospital „Sf. Ioan“, Galati; 3National Institute of Research-Development for Microbiology and Immunology „Cantacuzino“

Key words: Escherichia coli, virulence gene, PCR detection

urinary control. Growth of > 105 colony-forming units(CFU) per ml and the presence of at least 10 whiteblood cells per mm3 were considered as significant forthe presence of a UTI.

PCR for virulence factor-encoding genes

All isolates were also screened for five key viru-lence markers of extraintestinal pathogenic E. coli(ExPEC) isolates, including pap (P fimbriae), sfa/foc (Sand F1C fimbriae), afa (Dr-binding adhesins), hly(hemolysin) and cnf (cytotoxic necrotizing factor). Theprimers' sequences and the annealing temperatureshave been previously published (5). The PCR mixture(50µl) containing 10 µl of bacterial lysate, 200 µMdNTPs, 5 µl 10x PCR buffer, 1.25 U Taq DNA polyme-rase (Roche) and the primer set (30 pmol each of pap,sfa/foc, afa primers; 30 pmol each of hly primers; 30pmol each of cnf primers) was amplified in a thermalcycler MyCycler BioRad. The amplified products wereseparated in 1.5% agarose gels, and ethidium bro-mide-stained gels were visualized with an ultraviolettransilluminator for photographic imaging. E. coli J96and A30 strains were used as PCR positive controls. E.coli HB101 strain was used as PCR negative control.

RESULTS AND DISCUSSION

According to the PCR-based detection, among the54 E. coli urine isolates from children, 39 strains car-ried at least one of the targeted virulence determinants.The prevalence of these genes ranged from 9% for afato 55% for hly (Table 1). Almost all the PCR positivestrains of this collection possessed genes involved inthe biosynthesis of adhesion systems (37 strains). Thepresence of pap operon in more than half of the E. coli

strains isolated from children is proof of their patho-genic potential, especially if considering the impor-tance of P fimbriae for E. coli pyelonephritis. Epidemio-logic studies in adults and children over many years indiverse geographic locations have consistently demon-strated that these adhesins are present in nearly 100%of strains causing pyelonephritis (6). In human experi-ments, P fimbriae enhanced the early establishment ofbacteriuria (7).

S and F1C fimbriae have received little attention aspossible contributors to urovirulence (8, 9), and S fim-briae in particular have been regarded as pathogeneti-cally relevant principally in neonatal meningitis. In thisstudy, the sfa/foc positivity of the screened E. coli uroi-solates was not further stratified as to sfa versus foc, butits prevalence is greater compared to other publishedreports on E. coli UTI in children (10).

In the present study, a high prevalence of hly posi-tive strains was observed, and the presence of the genecorrelated with a hemolytic phenotype on blood agar(not shown). In a comparative study regarding the pre-valence of virulence factors in E. coli urinary isolatesfrom infants with and without bacteremia, the presen-ce of hly gene was seen as a useful positive predictionfactor for bacteremia (11). Thus, the hemolysin pro-duction may be of great importance when evaluatingthe clinical outcome of the patient .

Among the PCR positive strains of this E. coli col-lection, the co-occurrence of several determinants wasidentified in 30 strains (Table 2). The majority of thesestrains carried genes encoding adherence factors aswell as genes encoding toxins. All cnf positive strainswere also hly positive. It is noteworthy that 50% of thetested urinary isolates from children were J96-likestrains, suggesting the simultaneous carriage of thevirulence determinants on large blocks of chromoso-

42

FLOREA et al.

Virulence-associated genes No. (%) of positive strains (n=54)hly 30 (55)pap 29 (54)sfa/foc 24 (44)cnf 19 (35)afa 5 (9)

Gene combinationsdetected in E. coli uroisolates No. (%) of PCR positive strains (n=39)Genes associated with adhesins pap+afa 1Genes associated with adhesins + toxinspap+sfa/foc+ hly+cnf 12pap+sfa/foc+hly 7sfa/foc+hly+cnf 5pap+hly 3pap+hly+cnf 2

Table 1. Prevalence of virulence factor-ecoding genes in E. coli urinary strains isolated from children

Table 2. Co-occurrence of virulence genes in E. coli uroisolates from children

mal DNA known as „pathogenicity-associated-islands"(PAIs).

JOHNSON J.R. et al. proposed the designation ofExPEC for the highly specialized subset of the total E.coli population possessing extraintestinal pathogenicpotential due to their specific virulence factors (12),while MARRS et al. suggested that even separate UPEC(uropathogenic Escherichia coli) pathotypes exist (13).Considering as ExPEC the strains possessing at leasttwo of the virulence factors targeted in this study, 30urine cultures from children could be considered asExPEC positive. The PCR negative E. coli isolates couldbe indicative for identify children warranting anatomi-cal investigation.

The PCR approach used during the laboratory in-vestigation of this lot of children hospitalized for eitherurinary symptoms or systemic symptoms was very use-ful for identifying the intrinsic pathogenic potential ofE. coli uroisolates. In order to better assess the impor-tance of either the microorganism's virulence or thehost's susceptibility for the development of the UTIprocess, further studies on a greater number of E. coliurinary isolates from a well-defined population withdetailed epidemiologic and clinical information asso-ciated with each isolate are needed.

REFERENCES

1. Zorc J.J., Kiddoo D.A., Shaw K.N. Diagnosis and Manage-ment of Pediatric Urinary Tract Infections. Clin. Microbiol.Rev. 18: 417-422, 2005.

2. Ronald A. The etiology of urinary tract infection: traditionaland emerging pathogens. Am. J. Med. 113 (Suppl. 1A): 14-19, 2002.

3. Johnson, J. R. Virulence factors in Escherichia coli urinarytract infection. Clin. Microbiol. Rev. 4:80-128, 1991.

4. Hacker, J., G. Blum-Oehler, I. Muhldorfer, and H. Tschape.Pathogenicity islands of virulent bacteria: structure, func-tion, and impact on microbial evolution. Mol. Microbiol.23: 1089-1097, 1997.

5. Usein C.R., Damian M., Tatu-Chitoiu D., Capusa C.,Fagaras R., Tudorache D., Nica M., Le Bouguenec C.Prevalence of virulence genes in Escherichia coli strainsisolated from Romanian adult urinary tract infection cases.J. Cell. Mol. Microbiol. 5: 303-310, 2001.

6. Kallenius G, Mollby R, Svenson SB, Helin I, Hultberg H,Cedergren B, Winberg J. Occurrence of P-fimbriatedEscherichia coli in urinary tract infections. Lancet 2: 1369-1372, 1981.

7. Wullt B, Bergsten G, Connell H, Rollano P, Gebretsadik N,Hull R, Svanborg C. P fimbriae enhance the early establi-shment of Escherichia coli in the human urinary tract. MolMicrobiol 38: 456-464, 2000

8. Marre, R., J. Hacker, W. Henkel and W. Goebel. Contri-bution of cloned virulence factors from uropathogenic

Escherichia coli strains to nephropathogenicity in an expe-rimental rat pyelonephritis model. Infect. Immun. 54: 761-767, 1986.

9. Maslow, J. N., T. S. Whittam, C. F. Gilks, R. A. Wilson, M.E. Mulligan, K. S. Adams and R. D. Arbeit. Clonal rela-tionships among bloodstream isolates of Escherichia coli.Infect. Immun. 63: 2409-2417, 1995.

10. Arisoy M., Aysev D., Ekim M., Özel, D., Köse, S. K., Özsoy,E. D., Akar, N. Detection of virulence factors of Escherichiacoli from children by multiplex polymerase chain reaction.Internat. J. of Clin. Practice, Volume 60 (2): 170-173, 2006.

11. Bonacorsi S., Houdouin V., Mariani-Kurkdjian P., Mah-joub-Messai F., Bingen E. Comparative prevalence of viru-lence factors in Escherichia coli causing urinary tract infec-tion in male infants with and without bacteremia. J. Clin.Microbiol. 44: 1156-1158, 2006.

12. Johnson J.R., Russo T.A. Extraintestinal pathogenic Esche-richia coli (ExPEC): the „other bad E. coli“. J. Lab. Clin.Med. 139: 155-162, 2002.

13. Marrs C.F., Zhang L., Foxman B. Escherichia coli media-ted urinary tract infections: Are there distinct uropathoge-nic E. coli (UPEC) pathotypes? FEMS Microbiol. Lett. 252:183-190, 2005.

Virulence Characteristics of Escherichia coli Isolates From Children with Urinary Tract Infections

43

INTRODUCTION

In 1988 the World Health Organization (WHO)proposed to the Member States the worldwidepoliomyelitis eradication, based on maintaining highvaccination coverage (>80%) and on implementingeffective poliovirus infection surveillance systems.

Poliomyelitis is an acute paralytic disease causedby three distinct serotypes of poliovirus. Poliovirus*(PV) is a small, nonenveloped RNA virus of the ente-roviruses subgroup of the Picornaviridae family. Theresponse to poliovirus infection has been classifiedbased on the severity of clinical presentation. Of allpoliovirus infections less than 1% result in acute flac-cid paralysis (AFP), up to 95% are inapparent orasymptomatic, 4-8% consist of a minor illness (abor-tive poliomyelitis), 1-2% result in a nonparalyticaseptic meningitis (1).

The disease has been controlled by introductionand widespread use of formalin Inactivated Polio-virus Salk Vaccine (IPV ) in 1955 and Oral PoliovirusVaccine Sabin (OPV) in 1959 (2,3). For the eradica-tion of poliomyelitis the OPV was the vaccine of choicebecause it induces both a systemic immune responsewhich prevents the spread of poliovirus to the nerv-ous system and a mucosal immune response consis-ting of virus specific Ig A antibody secreted by theepithelial lining of the oropharyngeal and gastroin-testinal tract. The OPV consists of three live attenua-ted Sabin poliovirus strains obtained from wild strainsby sequential in vitro and in vivo passages.

The eradication program in most countries con-sists of high routine coverage with at least three dosesof oral poliovirus vaccine (OPV). The genetic insta-bility of OPV strains may lead to the emergence and

44

ABSTRACTUntil 2008 poliomyelitis was controlled in Romania by predominantly using Oral Poliovirus Vaccine Sabin(OPV); the alternative vaccination schedule (IPV formalin Inactivated Poliovirus Vaccine / OPV) will be imple-mented starting September 2008. The vaccination coverage with 4 doses of TOPV (trivalent oral polio vac-cine) in the first 14 months of life has been > 90 % since 1980. In Romania, the risk of the Vaccine-AssociatedParalytic Poliomyelitis cases (VAPP) decreased from less than 2 VAPP cases/year in the 1995-2006 interval to0 VAPP cases in 2007. The serological study was performed in 2006-2007 only in cases with pair serum sam-ples from 28 acute flaccid paralysis (AFP) cases (age = 3 months - 14 years) and from 45 facial paralysis (FP)cases (age =6 months - 4 years 9 months). A high level of vaccinal coverage was shown for all poliovirusserotypes: >95% in AFP serum samples investigated; and for FP serum samples investigated the levels of anti-bodies against poliovirus (PV) serotypes were 98% for PV type 1; 87% for PV type 2: and 89% for PV type 3.If the European region is polio free since 2002, the risk of wild PV importation from endemic region remainspresent. The laboratory capacity for the fast detection and molecular investigations of the emergence of thenew epidemic strains and a high level of population immunity must be maintained. A national seroprevalencestudy concerning all three PV serotypes must be performed.

THE MAINTAINING OF THE ACTIVE LABORATORY-BASED SURVEILLANCEOF THE ACUTE FLACCID PARALYSIS (AFP) CASES IN ROMANIA

IN THE FRAMEWORK OF THE STRATEGIC PLAN OF THE GLOBAL POLIO ERADICATION INITIATIVE

Anda Bãicuº1,2,3, Ana Persu1, Mariana Combiescu1 and A. Aubert-Combiescu1,2

1Cantacuzino Institute, Bucharest Romania; 2University of Medicine Bucharest;3Réseau d'Epidémiologie Clinique International Francophone Bucharest Romania

Key words: poliomyelitis, Oral Poliovirus Vaccine, IPV formalin Inactivated Poliovirus Vaccine, Vaccine-AssociatedParalytic Poliomyelitis

* The virion consists of a single stranded messenger - sense RNA genome of approximately 7500 nt. and 60 copies of each of thefour capsid proteins VP1 to VP4. The viral RNA contains 5' and 3' noncoding region (NCR) important for viral translation and repli-cation that flank along open reading frame (ORF) coding for the structural and non structural proteins. The strategy of the poliovirusRNA replication is common to all positive - stranded RNA viruses: the viral genome is transcribed into a complementary RNA - nega-tive strand, which in turn is used as template to synthesize new strands of genomic RNA. The synthesis of both strands is catalyzed by thevirus encoded RNA -dependent RNA polymerase of poliovirus but caused the error-prone by lacking proof-reading activity, resultingin a rapid accumulation of mutation upon PV replication (introduces nucleotide mutations with a mean frequency of about 10-4).

excretion into the environment of new variants ofpolioviruses differing from the original Sabin strains.Two natural evolution mechanisms are responsiblefor the emergence of new variants, nucleotide substi-tution (mutation) and molecular recombination (thesimultaneous administration of three poliovirus sero-types in the OPV facilitates intermolecular recombi-nation between the genomes of the three serotypes ofpolioviruses).

The major risks of OPV are the appearance ofthe Vaccine-Associated Paralytic Poliomyelitis cases(VAPP) and the development and circulation of patho-genic Vaccine Derived Polioviruses strains (VDPV).The incidence of VAPP was estimated at one case per750.000 doses for immunocompetent children recei-ving their first dose of TOPV (Trivalent OPV) (4).VDPVs resemble Wild Poliovirus strains (WPVs) bio-logically and differ from the Sabin vaccine-relatedpoliovirus isolates by having genetic properties con-sistent with prolonged replication or transmission.

The main objectives of the strategic plan of theGlobal Polio Eradication Initiative for 2004-2008 are:to implement immunization and surveillance activi-ties needed to interrupt wild poliovirus transmission;to achieve global certification of polio eradication; toprepare for eventually stopping the use of oral poliovaccines (5). The global incidence of polio associatedwith wild polioviruses (WPVs) was reduced from an350,000 cases in 1988 to 1,998 reported cases in2006, and the number of countries that have neversucceeded in interrupting WPV transmission droppedto four (Afghanistan, India, Nigeria and Pakistan) (6).

Although the European region is polio free since2002, the risk of importation from endemic regionremains present and a high level of population immu-nity must be maintained (7).

In Romania the national surveillance for paraliticpoliomyelitis began in 1949 and a comprehensivesurveillance system has been developed and main-tained since 1970, mainly for VAPP. The AFP sur-veillance was introduced since 1992 as recommen-ded by WHO. The reported incidence of paralytic po-liomyelitis decreasing from approximately 10 casesper 100 000 population in 1949 to 0,1 cases per 100000 population by the mid 1980. Between 1927-1960 the evolution of poliomyelitis was sporadic andepidemic (with outbreaks in 1927, 1946, 1953, 1957,1959). The latest outbreak of wild type poliovirusoccurred between November 1990 and April 1992with type 1 poliovirus (13 cases) and involved chil-dren from Roma communitis who were unvaccinatedor inadequately vaccinated. Four of 13 children werealso infected with HIV.

Until 2008 in Romania poliomyelitis was con-trolled by predominantly using OPV; the alternativevaccination schedule (IPV/OPV) will be implementedstarting September 2008. The vaccination coveragewith 4 doses of TOPV (trivalent oral polio vaccine) in

the first 14 months of life has been > 95 % since1980. The risk of recipient VAPP in Romania wasfound to be higher after the introduction of the OPVin 1961 than generally recorded in OPV-using coun-tries. This high rate was found to be associated withmultiple intramuscular injections during the incuba-tion period of the OPV strains (12). Measures to re-duce these simultaneous injections significantly lo-wered the VAPP rate (13). A reduction of parenteraltreatment in recipients of OPV and the change of theadministation schedule of oral polio vaccine from 2campaigns of two rounds, to throughout - the -yearvaccination since April 1995 determined a decreaseof the risk of VAPP with an average of less than 2VAPP cases/year in the 1995-2006 interval, and noVAPP case in 2007.

The surveillance system focuses on activesearching for acute flaccid paralysis AFP cases througha laboratory network from each of 42 counties, coor-dinated by the Ministry of Health, by the Institute ofPublic Health and by the National Poliovirus Laboratoryin Cantacuzino Institute (NPL). The Regional PolioLaboratory for this country is located in Pasteur Insti-tute Paris, France, and it is responsabile for intratypicdifferentiation and for confirmation as Sabin -like ornot Sabin-like for all PV strains isolated in Romania.

The objectives of our study were to make a com-parison between laboratory surveillance results in2001-2006 and 2007.

METHODS

The case investigation is conducted by the coun-ty epidemiologist and includes the collection of theclinical and epidemiologic information and samplescollection.

Between 2001-2007 the county laboratory sentsamples (stool samples, throat swab, etc) to the NPLfor virological investigations. Samples were processedaccording to a standard protocol for virus isolationand characterization at the NPL in the CantacuzinoInstitute, Bucharest, Romania. L20B (a geneticallyengineered mouse cell line expressing the humanpoliovirus receptor PVR and RD cells (derived from arhabdomyosarcoma) were inoculated with the sam-ples, as recommended by WHO for human entero-virus (HEV) detection (14-16). RD cells can be infectedby most enteroviruses, L20B cells are known to besensitive only to poliovirus. L20B and RD were grownin monolayers in Dulbecco's modified Eagle's medi-um supplemented with 10% foetal calf serum. Oncethe complete cytopathic effect (CPE) was obtained,the infected cells were harvested and kept frozen(-20°C). Typing of isolates was performed by neutrali-sation with pools of locally produced poliovirushorse antisera followed by confirmation with mono-type specific antisera.

The maintaining of the Active Laboratory-based surveillance of the acute flaccid paralysis (AFP) cases

45

The isolates were investigated by molecular me-thods: reverse transcription (RT), polymerase chainreaction (PCR) and restriction fragment length poly-morphism assays (RFLP) applied to two sequences ofthe viral genome which are located in 3D (6086nt -6516 nt ) and VP1-2A (2870 nt - 3648 nt) regions. Thetwo regions investigated with RT-PCR-RFLP assaywere chosen for the detection of recombinant strainsand for the confirmation of vaccinal origin of strainsand for their genotyping as well.

The study about the seroprevalence of antibo-dies to poliovirus was carried out in order to deter-mine the serological status against poliomyelitis. Inour serological study Acute -phase (AP) and conva-lescent-phase (CP) serum specimens have been testedfor neutralizing antibody to each of the three poliovirus.

Antibodies against poliovirus types 1,2,3 weredetermined with a microneutralization assay as pre-viosly described in our study (18 ) using 100 TCID50(100x 50% tissue culture infective dose) of challengevirus (Sabin strains ), according to the WHO - guide-lines (16) at the National Reference Center for Entero-viruses located in the Cantacuzino Institute Bucha-rest, Romania (NRCE-IC). The serum antibody titerwas the highest serum dilution that protected 50% ofcultures against 100 TCID50 of challenge virus. A se-rum sample was considered positive (indicating im-munity to poliomyelitis) if antibodies were present ata dilution 1:8.

RESULTS

In our study carried out between 2001-2006 inRomania in the NPL, samples from 430 cases (216AFP cases and 214 FP facial paralysis cases) and from2150 healthy contacts were investigated (17). 67 PVstrains were isolated from 21 AFP cases, 5 from FPcases and from 41 healthy contacts. In 2007 samplesfrom 22 AFP cases, 42 FP cases and 274 healthy con-tacts were investigated in the NPL, 2 PV strains wereisolated from healthy contacts and from FP cases. Allthe strains isolated in Romania between 2001-2007were Sabin - like with one exception, namely the strainisolated from an AFP case in Tulcea, Babadag county.From 21 AFP cases polio positive 7 cases were con-firmed as VAPP and one case as VDPV (Table 1).

The frequency of appearance and profiles of re-combinant genomes in 3D polimerase region forstrains investigated was the higest (44%) for Sabin3/Sabin 2 and the smallest (4%) for Sabin 1/Sabin 2.It is often reported that recombinant genomes frequen-tly occur among in OPV strains excreted by healthyvaccinated individuals, their contacts in the commu-nity, and in patients with VAPP. Genetic recombina-tion appears to be an integral part of poliovirus evo-lution.

The study in AFP, FP cases performed between2002-2005 on the seroprevalence of antibodiesagainst poliovirus types showed that the seropreva-

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BAICUS et al.

Table 1 - Epidemiological and laboratory data for acute flaccid paralysis AFP, facial paralysis (FP) cases and healthy polio positive contacts

lence of antibodiest against types 1 and 2 PV Sabinstrains was higher (>90%) than for type 3 PV Sabinstrains (85.7%). (18) The lower seroprevalence of anti-bodies against PV type 3 as compared to PV types 1and 2 was that most of the poliovirus strains isolatedbelonged to type 3 PV. The local conditions for fre-quent recombination events between type 3 PV andother serotypes were created.

Between 2006-2007 samples from 86 FP casesand 68 AFP cases were investigated in the National

Polio Laboratory in the Cantacuzino Institute. Howe-ver the serological study was performed only in caseswith pair serum samples from 28 AFP cases (age = 3months - 14 years) and from 45 FP cases (age =6months-4 years 9 months). A high level of vaccinalcoverage was shown for most poliovirus serotypes:for AFP serum samples investigated and for FP serumsamples the levels of antibodies were: 98% againstPV type 1, 87% against PV type 2 and 89% againsttype 3 (Table 2, Table 3).


47

DISCUSSION

In Romania the risk of VAPP decreased from anaverage of less than 2 VAPP cases/year in the 1995-2006 interval to 0 VAPP cases in 2007. Beginningwith September 2008 the combinated schedule IPV/OPV will be introduced in Romania. IPV elicits anti-bodies in the bloodstream, not in the intestines pre-venting paralysis. OPV induced intestinal immunity(limits the multiplication of the virus inside the in-testin and reduces fecal excretion) and serum immu-nity. The use of OPV can produce secondary immuni-zation of contacts through the spread of a vaccinevirus in stools. If IPV-vaccinated children are infectedby wild PV, they can become a source of infection iftheir antibody levels are not high enough to stopvirus excretion.

The epidemiological advantages of a vaccineswitch to IPV include the reduction of cVDPV, theelimination of VAPP cases and the discontinuation ofthe transmission of the vaccine virus.

When the global eradication of poliomyelitiswill be achieved, vaccination with OPV vaccine willbe stopped. The new recombinant enterovirus strainscould occupy the ecological niche taken by poliovirus.

In 2002 in Babadag, Tulcea county Romania wasisolated a PV type 1 strain from an AFP case (rroma

child - 5 months old not vaccinated against polio-myelitis ) and from 8 healthy contacts considered tobe at „risk“ (living in groups with low social and san-itary status, relatively low vaccination coverage 50%,because not all listed with family physicians). Afterantigenic and molecular analysis these isolates wereconfirmed as VDPV (recombinant Sabin 1 / Sabin 2 /Sabin 1) (19). Between 1 July and 15 November 2007in Romania there was an outbreak of aseptic menin-gitis. In temperate climates, enterovirus infections area more frequent occurrence in summer and autumn(20). In the NPL samples from 1101 patients withaseptic menigitis have been investigated. Virus isola-tion was performed from cerebrospinal fluid andstools. Nonpolio enterovirus isolates were obtainedfrom 376 patients samples; the majority of isolatedwere ECHO virus serotype 4 (151 samples) (21). Thedisease was mild and self-limiting with an averagehospital stay of few days. In order to study the gene-tic relationship of echovirus type 4 isolates will bemolecularly investigated by sequence analysis of aVP1-2A and 3D regions. Isolates from different geo-graphic regions will be compared to each other aswell as to prototype strain .

In countries where wild PVs were eradicated mostVDPV were isolated strains as OPV/HEV-C (HumanEnteroviruses Group C) recombinants.It was indica-

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BAICUS et al.

Table 2 - Immunity status against poliovirus type in acute flaccid paralysis (AFP) cases

ted that co-circulation of HEV-C and OPV strains isassociated with evolution by recombination and pro-bably contributed to the emergence of recombinantVDPVs in small human populations in some regions(8, 11, 22-25).

The surveillance of the co-circulation and evolu-tion of polio and non-polio enteroviruses must beincreased. Particular attention must be paid to theserological surveys at national level that are useful foridentifying groups with low-immunity that could beat risk of infection, to be maintaining of the nationalvaccinal coverage more 95% and the fast detection ofthe emergence of new epidemic strains.

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