Microbiological diagnosis of infectious keratitis

Conference (2013, March 16)

Capsular Gene Typing of Streptococcus agalactiae Compared to Serotyping by Latex Agglutination

in Journal of Clinical Microbiology (2012), 51(2), 503-507

We evaluated three different PCR-based capsular gene typing methods applied to 312 human and bovine Streptococcus agalac- tiae (group B Streptococcus [GBS]) isolates and compared the results to serotyping ... [more ▼]

We evaluated three different PCR-based capsular gene typing methods applied to 312 human and bovine Streptococcus agalac- tiae (group B Streptococcus [GBS]) isolates and compared the results to serotyping results obtained by latex agglutination. Among 281 human isolates 27% could not be typed by latex agglutination. All 312 isolates except 5 could be typed by the three PCR methods combined. Two of these methods were multiplex assays. Among the isolates that were typeable by both latex ag- glutination and capsular gene typing, 94% showed agreement between the two methods. However, each of the PCR methods showed limitations. One of the methods did not include all 10 recognized serotypes, one misidentified eight isolates of serotypes Ib and IV as serotype Ia, and one did not distinguish between serotypes VII and IX. For five isolates that showed aberrant pat- terns in the capsular gene typing, long-range PCR targeting the cps operon disclosed large insertions or deletions affecting the cps gene cluster. A sensitive flow cytometric assay based on serotype-specific antibodies applied to 76 selected isolates that were nontypeable by latex agglutination revealed that approximately one-half of these did express capsular polysaccharide. A proce- dure for convenient and reliable capsular gene typing to be included in epidemiological and surveillance studies of S. agalactiae is proposed. [less ▲]

Direct identification of bacteria from BacT/ALERT anaerobic positive blood cultures by MALDI-TOF MS: MALDI Sepsityper kit versus an in-house saponin method for bacterial extraction.

in Journal of Medical Microbiology (2012), 61

In cases of bacteraemia, a rapid species identification of the causal agent directly from positive blood culture broths could assist clinicians in the timely targeting of empirical antimicrobial therapy ... [more ▼]

In cases of bacteraemia, a rapid species identification of the causal agent directly from positive blood culture broths could assist clinicians in the timely targeting of empirical antimicrobial therapy. For this purpose, we evaluated the direct identification of micro-organisms from BacT/ALERT (bioMérieux) anaerobic positive blood cultures without charcoal using the Microflex matrix-assisted laser desorption/ionization (MALDI) time of flight MS (Bruker), after bacterial extraction by using two different methods: the MALDI Sepsityper kit (Bruker) and an in-house saponin lysis method. Bruker's recommended criteria for identification were expanded in this study, with acceptance of the species identification when the first three results with the best matches with the MALDI Biotyper database were identical, whatever the scores were. In total, 107 monobacterial cultures and six polymicrobial cultures from 77 different patients were included in this study. Among monomicrobial cultures, we identified up to the species level 67 and 66 % of bacteria with the MALDI Sepsityper kit and the saponin method, respectively. There was no significant difference between the two extraction methods. The direct species identification was particularly inconclusive for Gram-positive bacteria, as only 58 and 52 % of them were identified to the species level with the MALDI Sepsityper kit and the saponin method, respectively. Results for Gram-negative bacilli were better, with 82.5 and 90 % of correct identification to the species level with the MALDI Sepsityper kit and the saponin method, respectively. No misidentifications were given by the direct procedures when compared with identifications provided by the conventional method. Concerning the six polymicrobial blood cultures, whatever the extraction method used, a correct direct identification was only provided for one of the isolated bacteria on solid medium in all cases. The analysis of the time-to-result demonstrated a reduction in the turnaround time for identification ranging from 1 h 06 min to 24 h 44 min, when performing the blood culture direct identification in comparison with the conventional method, whatever the extraction method. [less ▲]

Phenotypical and genotypical surveillance of macrolide and lincosamide resistance in group B streptococcus in Belgium

in Program and Abstract of the 52nd Intersciences Conference on Antimicrobial Agents and Chemotherapy (2012, September)

Background: Constant increase of erythromycin (E) and clindamycin (C) resistance (R) has been observed worldwide among isolates of group B streptococci (GBS). In Belgium, through the 2000s, E R increased ... [more ▼]

Background: Constant increase of erythromycin (E) and clindamycin (C) resistance (R) has been observed worldwide among isolates of group B streptococci (GBS). In Belgium, through the 2000s, E R increased rapidly from 10% to up to 30%. Therefore phenotypical and molecular surveillance of E and C R has to be conducted. Methods: 275 clinical isolates (N1) were obtained from a Belgian surveillance for invasive GBS disease in newborns (59 isolates with 32 early- and 27 late-onset diseases) and adults (216 strains) during 2008 to 2011 and 53 isolates (N2) from vagino-rectal colonization in pregnant women in 2010. E and C MICs were determined by using Etest® (EUCAST interpretive criteria). Furthermore, for the E R isolates, the inducible (iMLS), constitutive (cMLS) and M phenotypes were assessed by a double disk diffusion test; the distribution of genes encoding RNA methylases and efflux pumps was investigated by PCR. Results: Of the N1 and N2 isolates, 92 (33.5%) and 15 (28.3%) were respectively R to E, with a higher rate among serotype V (p <0.001) and serotype IV (p <0.05). Among these 107 E-R isolates, 100 (93.5%) exhibited the MLS phenotype (R to E and CC): 73 were cMLS with E MIC50 >256 mg/L and 27 iMLS with E MIC50/MIC90 12/>256 mg/L. The M phenotype (R to E and S to C) was expressed by 7 (6.5%) of E R isolates with E MIC50/MIC90 4/12 mg/L. One colonizing strain presented a newly described resistance mechanism in GBS: the L phenotype (S to E and R to C) with a C MIC at 8 mg/L. For cMLS, the most common E R genotype was ermB (66%) (p <0.05) followed by ermTR (29%) and ermB+ermTR (5%). All iMLS isolates harbored an ermTR gene except 3 (2 with ermB, 1 with both ermB and ermTR); and all M phenotype were positive for mefA/B gene. Conclusions:1) In Belgium, by year 2010, prevalence of macrolides R in GBS exceeded 30%, 2) MLS R phenotypes (target-site modification) were the majority mechanism; M phenotype (efflux R mechanism) was also prevalent. 3) E and C susceptibility testing and surveillance are mandatory to guide prophylaxis and treatment of serious GBS infections in penicillin-allergic patients (at high risk for anaphylaxis) but also to identify emergence of newly acquired resistance mechanisms such as the L phenotype. [less ▲]

Acute cholecystitis with Listeria monocytogenes

in Acta Clinica Belgica (2012), 67(4), 295-297

Listeriosis, an opportunistic food-borne disease caused by Listeria monocytogenes, is infrequent and occurs preferentially in patients at the extremes of age, during pregnancy or in immunocompromised ... [more ▼]

Listeriosis, an opportunistic food-borne disease caused by Listeria monocytogenes, is infrequent and occurs preferentially in patients at the extremes of age, during pregnancy or in immunocompromised hosts. Most common manifestations are maternofoetal and neonatal infections, severe invasive presentations such as bacteraemia with or without central nervous system symptoms occuring preferentially in immunosuppressed patients and self-limited gastro-enteritis affecting healthy individuals. Exceptionally, focal infections such as cholecystitis are described. We report here a case of acute cholecystitis caused by Listeria monocytogenes in an 82-year-old woman. Thanks to a successful treatment: cholecystectomy and antimicrobial therapy (amoxicillin plus clavulanic acid), the patient soon recovered. This case-report provides an opportunity to review the current literature concerning the association of Listeria monocytogenes and cholecystitis. [less ▲]

Proportion of extended-spectrum ß-lactamase-producing Enterobacteriaceae in community setting in Ngaoundere, Cameroon

in BMC Infectious Diseases (2012), 12

BACKGROUND: There is no information regarding the resistance mechanisms of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in community setting in Cameroon. The current study aimed to ... [more ▼]

BACKGROUND: There is no information regarding the resistance mechanisms of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in community setting in Cameroon. The current study aimed to determine the proportion of ESBLs in Enterobacteriaceae isolated in the community and to analyse some risk factors associated with ESBL carriage. METHODS: Faecal samples were collected from 208 different outpatients and 150 healthy student volunteers between 3 January and 3 April 2009. Enterobacterial isolates resistant to third-generation cephalosporins were screened for ESBL production by the double-disk synergy test. Presumptive ESBL-producing isolates with positive synergy test were identified by Mass Spectrometry using the BioTyper MALDI-TOF. For such ESBL positive isolates, antibiotic susceptibility was determined by the Vitek 2 system. PCR and sequencing were performed for the detection of different types of ESBL genes in presumptive ESBL-producing isolates. Statistical methods were used for the univariate calculation of risk factors. RESULTS: During the study period, a total of 358 faecal samples were analysed; 58 of such samples (16%) showed an ESBL phenotype and were confirmed by PCR. The proportion of ESBL producers in faecal carriage was statistically different between outpatients and student volunteers (23.1% vs. 6.7%: p < 0.000). According to a univariate analysis, previous use of antibiotics (ciprofloxacin) appeared to be a risk factor for ESBL carriage (p < 0.05).Escherichia coli was the species most frequently isolated among the ESBL producers in outpatients (66.7%) and student volunteers (90%). Isolates showed additional resistance to gentamicin, ciprofloxacin and trimethoprim/sulfamethoxazole but none of them was resistant to temocillin, amikacin or meropenem. Most of the strains (97%) produced a CTX-M group 1 enzymes [CTX-M-15 (98%) or CTX-M-1 (2%)] and the remaining strains produced SHV-12 enzyme (3%). CONCLUSIONS: The use of drugs such as amoxicillin, ciprofloxacin and trimethoprim/sulfamethoxazole does not seem appropriate for empirical treatment because of emerging resistance. The implementation in Cameroon or in other African countries of methods of screening ESBL-producing organisms in routine laboratories is of great importance in order for us to offer patients appropriate treatment and for infection control efforts to succeed. [less ▲]

Typage des souches de Norovirus circulant dans les populations symptomatiques et asymptomatiques au Burkina Faso

Poster (2012)

Appartenant à la famille des Caliciviridae, genre Norovirus, les norovirus (NoV) sont des virus non enveloppés dont le génome est composé d’un ARN monocaténaire de polarité positive ... [more ▼]

Appartenant à la famille des Caliciviridae, genre Norovirus, les norovirus (NoV) sont des virus non enveloppés dont le génome est composé d’un ARN monocaténaire de polarité positive d’approximativement 7,5 kb. Les NoV infectent l’homme chez qui ils représentent au niveaumondial un agent majeur de gastroentérites épidémiques, d’origine souvent alimentaire mais aussi sporadique, et ce, toutes classes d'âges confondues. Les souches humaines sont classées génétiquement dans différents génotypes au sein de trois des cinq génogroupes, nommés (G) I, II et IV, composant le genre Norovirus. La voie de transmission des NoV est féco-orale. Les NoV sont très résistants dans l’environnement et la dose infectieuse est faible. Dans la population humaine, une grande diversité de souches appartenant principalement aux G I et II co-circulent. Parmi ces souches, le génotype Lordsdale (GII-4) est prédominant dans les épidémies actuelles, notamment lorsqu'une transmission de personne à personne est incriminée, alors que les souches du G I semblent plus fréquemment rapportées au cours des épidémies d’origine environnementale, comme celles liées à la consommation de fruits de mer. Si de nombreuses études d'épidémiologie moléculaire concernant ces virus ont été réalisées dans les pays industrialisés, les données sont par contre manquantes ou ténues pour bien des pays non industrialisés, et en particulier africains. Au cours d'une étude épidémiologique réalisée à Bobo Dioulasso au Burkina Faso et portant sur la prévalence des NoV dans les échantillons de selles de patients présentant ou non des symptômes de gastro-entérite, les souches détectées ont été quantifiées, leur génogroupe a été déterminé et pour certaines d'entre elles le génotype a été précisé. Quatre cent cinquante trois patients ont été prélevés, dont 319 présentant des symptômes diarrhéiques et 134 sujets témoins ne présentant pas de symptomatologie digestive. La détection des NoV et la quantification des charges virales excrétées ont été effectuées sur tous les échantillons par RT-PCR en temps réel permettant de discriminer les souches appartenant aux G I ou II. Une RT-PCR conventionnelle visant les régions de la polymérase (ORF1 du virus) ou de la capside (ORF2) a ensuite été réalisée sur une partie des échantillons détectés positifs en vue du séquençage de ces régions. Les relations phylogénétiques des souches circulant dans la population du Burkina Faso aux souches de référence ont aussi été inférées. Les résultats de RT-PCR en temps réel ont permis de mettre en évidence que les prévalences apparentes de l'infection par les NoV sont similaires dans les populations symptomatique et asymptomatique : une détection moléculaire de NoV chez 67 patients présentant de la diarrhée (21,0 %) et chez 31 des sujets témoins (23,1 %) a pu être observée. Les génotypes circulant détectés sont très variés dans les deux génogroupes, avec une proportion assez surprenante de NoV appartenant au G I. Université polytechnique de Bobo-Dioulasso, Institut supérieur des Sciences de la Santé (INSSA), Bobo-Dioulasso, Burkina Faso. Cette étude a permis de préciser l'épidémiologie moléculaire des souches de NoV circulant dans un pays représentatif de l'Afrique de l'Ouest. Elle a également montré que des individus asymptomatiques pourraient jouer un rôle assez important de réservoir du virus. Elle souligne enfin que, malgré le fait que les souches GII, et en particulier celles de génotype GII.4, soient à l'heure actuelle rapportées majoritairement au niveau mondial, les souches G I doivent être excrétées en égale proportion dans l'environnement. L'origine épidémiologique de la différence entre les prévalences apparentes des infections par les souches de GI et de GII, bien que partiellement expliquée par les différences de sensibilité génétique et d'immunité de population, reste donc à élucider. Remerciements: à la fondation A. Seghers, au Centre de Coopération au Développement de l'Université de Liège, à R. Boreux (assistance technique), aux membres du laboratoire du CMA de Dô et aux agents de santé de Bobo-Dioulasso (Burkina-Faso). [less ▲]

Group B streptococci, a European perspective with results of the DEVANI project

in DUCOFFRE, GENEVIèVE (Ed.) Abstract book of 27th Annual Meeting "Diagnostic et surveillance des maladies infectieuses" (2011, November)

In 2011, neonatal group B streptococcal (GBS) diseases remain a global public health concern. Where consensus guidelines to detect and treat intrapartum women with GBS colonization have been widely ... [more ▼]

In 2011, neonatal group B streptococcal (GBS) diseases remain a global public health concern. Where consensus guidelines to detect and treat intrapartum women with GBS colonization have been widely adopted, incidence of neonatal early onset disease (EOD) has dramatically declined, however despite preventive strategies cases still occur. The strategy was not expected to prevent all cases and there are challenges and limitations to this preventive approach. The best strategy for European countries is still a matter of debate and intrapartum antimicrobial prophylaxis (IAP) is not widely recommended. To adopt the best preventive strategy, we first need better data assessing more accurately the true burden of GBS diseases in the different countries. Furthermore, as the current screening-based strategy for prevention is highly effective but imperfect, given the challenges, limitations and potential complications of maternal IAP, a new approach is still needed. Maternal immunization against GBS is an attractive alternative for the prevention of not only neonatal diseases but also stillbirths and maternal diseases. Vaccines against GBS may likely become the most effective and sustainable long-term preventive strategy. But the development of vaccines with global relevance has been hampered by changes in the distribution of GBS serotypes of strains causing diseases over time and in different parts of the world. A multivalent vaccine to cover against the more prevalent serotypes suitable for European populations might not be suitable for Asian or African populations. To overcome type-specificity, new developments target vaccines based on conserved surface antigenic proteins, such as Sip protein located at the cell surface of all GBS and on immunogenic proteins from GBS pili. A pilus-based GBS vaccine is appealing and could become a globally relevant reality. The DEVANI (DEsign of a Vaccine Against Neonatal Infections) programme funded through the European Commission Seventh Framework was launched on 1 January 2008 with the key objective being the assessment of European GBS epidemiology to facilitate the design of a new vaccine that will confer neonatal immunity through a durable maternal immune response. A major component was to undertake pan European surveillance of maternal colonisation, maternal GBS antibody responses and neonatal diseases in eight European countries. Through 2009 and 2010, all Belgian laboratories sending any neonatal GBS invasive isolate to the National Reference Centre for GBS were invited to bring their contribution to this project. Belgium, Bulgaria, Czech Republic, Denmark, Germany, Italy, Spain and the United Kingdom established specific GBS screening studies during 2008/10. Maternal vaginal/rectal swabs and sera were taken between 34-37 weeks gestation and processed using a standardised microbiological screening protocol. Samples from neonatal cases were processed using local procedures. For each pregnant woman and each case of GBS neonatal disease, standardized case report forms were filled. GBS isolates were characterised using standardised serological and molecular typing methods for detection of all ten GBS capsular polysaccharide types (Ia to IX). Furthermore all the collected isolates were screened by multiplex PCR and FACS analysis to evaluate respectively gene presence and surface-exposure of pili. And clonal analysis of these isolates was performed using multi-locus sequence typing (MLST). The main microbiological results of this pan European surveillance are following. Carriage rates among pregnant women in all countries ranged from 8% to 26%. The most common GBS capsular types were III (33%), Ia (25%) and V (8%). Among GBS from EOD, the major serotypes were III (43%), V (21%) and Ia (18%). In contrast among GBS isolated from neonatal late onset disease (LOD), serotype III was highly predominant (80.6%) followed mainly by Ia (12.5%). Analysis of the pattern of pili genes showed that all isolates contained at least one gene coding for pili. The most common gene patterns found were PI-2a alone, PI 1+2a and PI 1+2b, while the PI-2b gene alone was very rare. The most prominent result was that a majority of isolates from neonatal infections carried the PI-1+2b gene pattern, while the most common pattern among pregnant women was PI-1+2a. Most of analyzed strains express at least one pilus on their surface. The clonal analysis showed that 66 sequence types were found to belong to nine clonal complexes (CC). Among these nine CCs, five were prevailing and covered 92 % of GBS isolates tested. The GBS population in pregnant women was found to be more heterogeneous than the GBS isolated from neonatal infection cases. Among neonatal isolates, the most frequent CC was CC17 (43 %) known as a highly virulent clone. Among participating countries, there were no significant differences in the occurrence of clonal complexes. The analysis of the levels of specific antibodies as surrogate markers of protection is still ongoing. More detailed and additional results as the main conclusions will be presented. [less ▲]

NEONATAL INVASIVE GROUP B STREPTOCOCCAL (GBS) INFECTIONS IN EUROPE

Poster (2011, September)

Objectives: To describe clinical characteristics and capsular type of GBS isolates responsible of invasive infections in infants from Belgium, Bulgaria, Czech-Republic, Denmark, Germany, Italy, Spain and ... [more ▼]

Objectives: To describe clinical characteristics and capsular type of GBS isolates responsible of invasive infections in infants from Belgium, Bulgaria, Czech-Republic, Denmark, Germany, Italy, Spain and United Kingdom, representing one of the main objectives of the DEVANI (DEsign of a Vaccine Against Neonatal Infections) project. Methods: Surveillance of invasive GBS infections in infants was performed from mid-2008 through December 2010. For each case, a standardized case report form was filled. Samples from cases were processed using local procedures. GBS isolates were characterised in national central labs using standardised type-specific (Ia, Ib-IX) latex agglutination and molecular typing methods. Results: Data on 188 infants with invasive infection were analysed: 144 (60.6%) early onset diseases (EOD) and 74 (39.4%) late onset diseases (LOD). In EOD, mean/median ages at onset were 14/0 hours and the male:female ratio was 1.25. The predominant manifestation at onset was respiratory distress (42% cases); 83% cases were associated with sepsis/bacteremia, 15% with pneumonia and 6% with meningitis. Late-prenatal screening cultures were obtained from 51% of cases’ mothers and only half of these were positive for GBS. Non-elective C-section, intrapartum fever and rupture of membrane (>18h) were more frequent in EO-cases’ mothers versus healthy babies’ GBS-positive mothers. The major serotypes were III (43%), V (21%) and Ia (18%). In LOD, mean/median ages at onset were 42/34 days and the male:female ratio was 0.9. The predominant characteristic at onset was fever (62% cases); 70% cases were associated with sepsis and 30% with meningitis. Very rare manifestations were osteomyelitis and cellulitis. Serotype III was highly predominant (80.6%) followed mainly by Ia (12.5%). Death rates were 4.7/1.5% in EOD/LOD. Conclusions: Clinical presentations were associated with age at onset of infection. Serotype III predominated in neonatal infections. Prenatal screening was not universal neither sensitive. Study funded through the European Commission Seventh Framework. [less ▲]

Severity of ICU-acquired pneumonia according to infectious microorganisms

in Intensive Care Medicine (2011), 37(7), 1128-35