Effect of clinical spectrum, inoculum size and physician characteristics on sensitivity of a rapid antigen detection test for group A streptococcal pharyngitis.

We aimed to assess the independent effect of clinical spectrum, bacterial inoculum size and physician characteristics on the sensitivity of a rapid antigen detection test (RADT) for group A streptococcus (GAS) in children. Double throat swabs were collected from 1,482 children with pharyngitis and 294 asymptomatic children in a French prospective, office-based, multicenter (n = 17) study, from October 2009 to May 2011. Patient- and physician-level factors potentially affecting RADT sensitivity were studied by univariate and multivariate multilevel analysis, with laboratory throat culture as the reference test. In children with pharyngitis and asymptomatic children, the prevalence of GAS was 38 % (95 % confidence interval 36-41 %) and 11 % (7-14 %), respectively. Overall, RADT sensitivity was 87 % (84-90 %). On stratified and multivariate multilevel analysis, RADT sensitivity was higher for children with pharyngitis than asymptomatic children (89 % vs. 41 %), children <9 than ≥ 9 years old (88 % vs. 79 %) and those with heavy than light inoculum (94 % vs. 53 %). RADT sensitivity was influenced by the physician performing the test (range 56-96 %, p = 0.01) and was higher for physicians with hospital-based clinical activity in addition to office-based practice (adjusted odds ratio 3.4 [95 % confidence interval 1.9-6.3], p < 0.001); inter-physician variations in RADT sensitivity were largely explained by this variable (proportional change in variance >99 %). The sensitivity of the RADT is independently affected by patient- and physician-level factors. Physicians who base their diagnosis of GAS pharyngitis on the results of a RADT alone should consider diagnostic accuracy monitoring and adequate training when needed.

Rapid, simple multi-locus variable number tandem repeat analysis: a reliable tool for Klebsiella pneumoniae outbreak screening.

BACKGROUND: Klebsiella pneumoniae causing nosocomial infections is increasingly multi-drug-resistant. Rapid and efficient typing tools are required for monitoring. AIM: To assess a simple, rapid (<5 h) multiplex polymerase chain reaction method based on multi-locus variable number tandem repeat analysis (MLVA) as a screening tool to determine whether or not K. pneumoniae strains are related. METHODS: The global discriminatory power of the method was assessed on 72 unrelated K. pneumoniae isolates, including community carriage isolates, highly virulent strains causing liver abscess, and extended-spectrum beta-lactamase- and carbapenemase-producing strains. Suspected related strains from a suspected outbreak and a relapsed meningitis case were also studied. MLVA results were compared with whole-genome sequencing (WGS) analysis and multi-locus sequence typing (MLST). FINDINGS: MLVA and MLST had similar discriminatory power, each distinguishing 54 profiles among the 72 unrelated isolates (Hunter-Gaston index 0.989). Each strain belonging to one sequence type (ST) or ST complex had its own MLVA type, with few exceptions. Two strains of ST268 and ST1119 shared the same MLVA profile, and two unrelated strains of ST307, ST86, ST45 and ST37 exhibited two different MLVA types each. Moreover, investigation of seven grouped cases of K. pneumoniae neonatal sepsis pointed to strong suspicion of a common source for five isolates, while two isolates with a different MLVA profile were excluded from this cluster. CONCLUSION: The MLVA approach is a useful, rapid and reliable tool for epidemiological investigation requiring only basic molecular biology equipment, and permits identification of sporadic isolates that are not part of an outbreak. However, analysis of strains sharing the same MLVA type by a highly discriminatory technique, such as WGS, remains necessary.

Identification of group A streptococcal emm types commonly associated with invasive infections and antimicrobial resistance by the use of multiplex PCR and high-resolution melting analysis.

M/emm typing, based either on serotyping of the M protein or on sequencing of the emm gene, is a major tool for epidemiological studies of group A streptococci (GAS). In order to simplify M/emm typing, we designed two multiplex polymerase chain reaction (PCR) formats capable of identifying the most frequent GAS M/emm types involved in invasive infections and antimicrobial resistance. A heptaplex PCR procedure was first developed in a conventional format coupled with gel electrophoresis to identify emm types 1, 3, 4, 6, 12, 28, and 89, based on the size of the amplification products. The other method, designed to identify the same seven emm types, together with emm11, was based on a real-time PCR format coupled with high-resolution melting (HRM) analysis, allowing the rapid typing of large strain collections.

Epidemiological investigation of Pseudomonas aeruginosa isolates including multidrug-resistant serogroup O12 isolates, by use of a rapid and simplified multiple-locus variable-number of tandem repeats analysis and whole genome sequencing.

BACKGROUND: Clustered cases of Pseudomonas aeruginosa infection in immunocompromised patients' wards require rapid characterization of a potential epidemic to guide investigations and identify the potential source of contamination. AIM: To design and evaluate a rapid and simple typing method for P. aeruginosa in comparison to whole genome sequencing (WGS). METHODS: A simplified polymerase chain reaction based on multiple-locus variable-number of tandem repeats analysis (MLVA) was designed and used to investigate cases of P. aeruginosa infection and colonization in a paediatric haematology department. The method was compared to WGS by using the Illumina method. FINDINGS: On the 17 isolates recovered from 15 children (eight from blood cultures, three from urinary tract infections, one from sputum and five stool isolates), MLVA distinguished 10 different profiles, and seven isolates from six children shared the same profile. Analysis by WGS revealed that these seven isolates belonged to sequence type ST111 and serotype O12, allowing at least three different genotypes to be distinguished among them. Five environmental strains had three MLVA profiles; one was shared with a clinical isolate but WGS excluded any relationship. CONCLUSION: The simplified and inexpensive MLVA method enabled the exclusion, in less than 5 h, of most of the unrelated isolates and thus to focus investigations on a small number of cases, whereas WGS, taking several days of work, drew definitive conclusions concerning the outbreak and the genetic relationships of the ST111 isolates circulating in the department. We conclude that sequential use of both methods is the optimal strategy to investigate clustered cases of P. aeruginosa infections.

Epidemiology of invasive Streptococcus pyogenes infections in France in 2007.

Invasive group A streptococcal (GAS) infections cause significant morbidity and mortality. A national survey was initiated to assess the burden of invasive GAS infections in France, describe their clinical characteristics, and assess the molecular characteristics of GAS strains responsible for these infections. The survey was conducted in 194 hospitals, accounting for 51% of acute care hospital admissions in France. Clinical data, predisposing factors, and demographic data were obtained, and all GAS isolates were emm sequence typed. We identified 664 cases of invasive GAS infections, with an annual incidence of 3.1 per 100,000 population. The case-fatality ratio was 14% and rose to 43% in the case of streptococcal toxic shock syndrome. Bacteremia without identified focus (22%) and skin/soft tissue infections (30%) were the most frequent clinical presentations. Necrotizing fasciitis was frequent in adults (18%) and uncommon in children (3%). The 3 predominant emm types were emm1, emm89, and emm28, accounting for 33%, 16%, and 10% of GAS isolates, respectively. The emm1 type was associated with fatal outcomes and was more frequent in children than in adults. Six clusters of cases were identified, with each cluster involving 2 invasive cases due to GAS strains which shared identical GAS emm sequence types. Four clusters of cases involved eight postpartum infections, one family cluster involved a mother and child, and one cluster involved two patients in a nursing home. Invasive GAS infection is one of the most severe bacterial diseases in France, particularly in persons aged ≥ 50 years or when associated with toxic shock syndrome.

Invasive group A streptococcal infection in children: clinical manifestations and molecular characterization in a French pediatric tertiary care center.

Invasive group A streptococcal (GAS) infections have a broad and evolving clinical spectrum, associated with various GAS genotypes and/or virulence factors that are only poorly described in children. We aimed to assess the clinical and molecular characteristics of invasive GAS infections in 28 children admitted from 2000 to 2007 at a large French pediatric tertiary care center. The GAS isolates were characterized molecularly by emm-typing and by the determination of the main virulence factors: speA, speB, speC, smeZ-1, ssa, sic, and silC. The median age of the children was 2.9 years. Osteoarticular infection (OAI) was the main clinical manifestation (n=15/28, 53%). emm-1 predominated (n=10/28), followed by emm-12, 3, and 4. No significant correlation was found between emm type and clinical manifestations, but emm-1 predominated in cases of OAI (n=7/15) and was associated with speA, speB, smeZ-1, and sic virulence factor genes. In this pediatric study, we describe a predominance of OAI associated with emm-1 GAS. Further larger international pediatric studies, including host immunity evaluation, are needed in order to better assess the pathogenesis of GAS infection in children.

Subtyping of emm1 group A Streptococci causing invasive infections in France.

By combining PCR amplification of toxin-encoding genes and sic gene sequencing, we distinguished 24 genotypes among 47 M/emm1 group A streptococci isolated from children and adults in France in 9 cases of infection comprising four clusters and 38 unrelated invasive infection cases used as controls.

[Clinical outcome and bacterial characteristics of 99 Escherichia coli meningitis in young infants]. / Méningite à Escherichia coli de l'enfant : analyse descriptive clinique et microbiologique de 99 cas.

OBJECTIVE: To conduct a descriptive analysis of clinical, biological and prognostic aspects of Escherichia coli meningitis in young infants. METHODS: Clinical and biological data on young infants diagnosed with neonatal E. coli meningitis (NECM) between 1988 and 2004 were collected retrospectively and analyzed with respect to the isolates'phenotypic and genotypic characteristics. The molecular analyses focused on the phylogenetic group, the sequence-O-type, and genetic virulence traits. The virulence of lethal strains was tested in a newborn rat meningitis model. RESULTS: The median age of the 99 children analyzed was 10 days (0 to 90 days), and 83 of the patients were newborns. Thirty-three children were premature. Hyper- or hypothermia was the most frequent clinical sign at admission. Intercurrent urinary tract infection was present in 28% of cases, all over 6 days of age. 81% of blood cultures were positive. The CSF cytology was abnormal in 97% of cases. Twelve hours after admission, 34% of infants were transferred to intensive care. One-third of transfontanellar ultrasound scans done on admission were abnormal. CSH sterilization was slow in 15 % of cases, despite appropriate antibiotic therapy. The use of ciprofloxacin was associated with more rapid CSF sterilization (94 % vs 77 %, p=0.03). Six children relapsed. The average follow-up was eight months, and 21 % of children had sequelae. The case lethality rate was 14%. Fatal outcome was associated with signs of septic shock (57% vs 3%, p<10(-4)) and neurological failure (76% vs 19%, p<10(-4)) within the first 24 hours, and with abnormalities on the first ultrasound scan (63% vs 27%, p=0.03). The risk of death was higher among children infected by strains belonging to unusual sequence-O-types (50% vs 18%, p=0.01), which harbored fewer virulence factors (4.8 vs 5.9, p<10(-4)). Only aerobactin was less frequent in lethal strains (71 % vs 94%, p=0.02). Strains belonging to unusual sequence-O-types and that were lethal in the animal model induced a significantly lower level of bacteremia than strains belonging to frequent sequence-O-types (p<0.001). CONCLUSION: E. coli meningitis remains highly lethal in infants. Clinical and molecular analyses showed a link between lethality and infrequent sequence-O-types. The avirulence of these strains in animal models suggests that fatal outcome could be due to host susceptibility more than to strain virulence.

Antimicrobial susceptibility of 136 Escherichia coli isolates from cases of neonatal meningitis and relationship with virulence.

The susceptibility of 136 Escherichia coli isolates from cases of neonatal meningitis to amoxycillin, ceftriaxone, nalidixic acid, ciprofloxacin and gentamicin was determined in relation to the carriage of virulence factors and phylogenetic group. Only amoxycillin and nalidixic acid resistance was observed (40% and 3%, respectively). Nalidixic acid resistance alone was associated with non-virulent phylogenetic group A (50% vs. 6% of susceptible isolates; p 0.03). No difference in virulence was observed between two representative nalidixic acid-susceptible virulent group B2 isolates and their nalidixic acid-resistant derivatives in a rat model of neonatal meningitis, suggesting that nalidixic acid resistance does not affect the virulence of E. coli strains causing meningitis.

Phylogenetic groups and virulence factors of Escherichia coli strains causing pyelonephritis in children with and without urinary tract abnormalities.

Escherichia coli isolates causing acute pyelonephritis in 93 children (25% with urinary tract abnormalities) were tested for nine virulence factors (papC, papGII, papGIII, sfa/foc, hlyC, cnf1, iucC, fyuA and iroN) and their phylogenetic groups were determined. Isolates lacking papGII were more frequent among patients with urinary tract abnormalities (58% vs. 10%, p 0.0003), as were non-virulent phylogenetic group A isolates (25% vs. 5%, p 0.043). Pyelonephritis caused by less virulent E. coli strains was more frequent among patients with significant urinary tract abnormalities. Further studies are required to determine whether screening for E. coli virulence factors may help to identify children warranting anatomical investigations.

First description of DHA-1 ampCbeta-lactamase in Proteus mirabilis.

This report describes the first occurrence of the DHA-1 ampCbeta-lactamase gene in Proteus mirabilis. The organism was isolated from the vaginal flora of a pregnant woman in a French hospital. The DHA-1 beta-lactamase gene was identified on the basis of phenotypic characteristics, PCR amplification and sequencing. Antagonism between cefoxitin and the other cephalosporins suggested inducible production of the DHA-1 enzyme.

Group B streptococcus neonatal invasive infections, France 2007-2012.

Streptococcus agalactiae (group B streptococcus (GBS)) is the leading cause of invasive infections among newborns in industrialized countries, with two described syndromes: early-onset disease (EOD) and late-onset disease (LOD). Since the introduction in many countries of intrapartum antibioprophylaxis (IAP), the incidence of EOD has dramatically decreased, whereas that of LOD remains unchanged. We describe the clinical and bacteriological characteristics of 438 GBS neonatal invasive infections notified to the French National Reference Centre for Streptococci in France from 2007 to 2012. Clinical data were retrieved from hospitalization reports or questionnaires. Capsular type, assignment to the hypervirulent clonal complex (CC)17 and antibiotic susceptibility profiles were determined. One hundred and seventy-four (39.7%) and 264 (60.3%) isolates were responsible for EOD, including death in utero, and LOD, respectively. EOD was associated with bacteraemia (n = 103, 61%) and LOD with meningitis (n = 145, 55%). EOD was mainly due to capsular polysaccharide (CPS) III isolates (n = 99, 57%) and CPS Ia isolates (n = 40, 23%), and CPS III isolates were responsible for 80% (n = 211) of LOD cases. CC17 accounted for 80% (n = 121) of CPS III isolates responsible for meningitis (n = 151; total cases of meningitis, 188). Bad outcome risk factors were low gestational age and low birthweight. LOD represents almost 60% of cases of neonatal GBS disease in France and other countries in which IAP has been implemented. This observation reinforces the need to develop new prevention strategies targeting CC17, which is predominant in GBS neonatal infections.

Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing.

PCR-ribotying, a typing method based on polymorphism in the 16S-23S intergenic spacer region, has been recently used to investigate outbreaks due to Clostridium difficile. However, this method generates bands of high and close molecular masses which are difficult to separate on agarose gel electrophoresis. To improve reading of banding patterns of PCR-ribotyping applied to C. difficile, a partial sequencing of the rRNA genes (16S and 23S) and intergenic spacer region has been performed, then a new set of primers located closer to the intergenic spacer region has been defined. The new PCR gave reproducible patterns of bands easy to separate on agarose gel electrophoresis. Each of the 10 serogroups and 11 subgroups of serogroup A produced a different pattern. This typing method has evidenced major qualities such as easiness, rapidity and reproducibility. However, its discriminatory power has to be evaluated to validate its importance as a typing tool for C. difficile.

[Anomalies of the membrane receptors of blood phagocytic cells in nosocomial pulmonary infections]. / Anomalies des récepteurs membranaires des cellules phagocytaires sanguines au cours d'infections pulmonaires nosocomiales.

Patients in intensive care units have frequent and severe opportunistic bacterial pneumoniae, even if they were previously free from respiratory disease. A search was made in these patients for possible immune deficiencies. Granulocyte and monocyte phagocytic activities were studied separately in 17 patients with bacterial bronchopneumonia (male: 13, female: 4; age: 41 +/- 5 yr). The ability of three types of particles (opsonized zymosan, immunoglobulin coated and glutaraldehyde-treated sheep red cells) to trigger ingestion was measured. Cells were tested either in normal AB serum or in the presence of patient's serum. A substantial 40% decrease of the fraction of granulocytes ingesting zymosan was found in our experimental conditions (p less than 0.001). Activity with the other particles was not significantly altered. Patients' sera were at least as efficient as a pool of normal sera in opsonizing zymosan. Furthermore, no phagocytic inhibitor was found in the patients tested. Underlying mechanisms for these abnormalities remain unknown, but a better understanding of the aetiology of the altered bactericidal function of phagocytic cells is required before suggesting immunomodulating treatments.

[Lack of prognostic value of the determination of 3 serum proteins during the acute phase of brain injury]. / Absence de valeur pronostique du dosage de trois protéines sériques à la phase aiguë du traumatisme crânien.

In has been suggested that monitoring of blood levels of serum proteins could be of value in order to assess the prognosis of trauma patients. A study was set up to assess the value of albuminaemia, prealbuminaemia, transferrin and ceruleoplasmin levels in intensive care unit patients admitted after multiple trauma including head injury. This study involved 43 patients (31 men and 12 women), with a mean age of 26 years (range: 15 and 71 years). Two patients had extradural haematoma. The other patients had brain contusion associated with extradural haematoma (5 cases), subdural haematoma (1 case) and open brain wound (1 case). All these patients were sedated with penthiobarbital, paralysed with pancuronium bromide and mechanically ventilated. Serum protein levels were measured on the first and eighth days of the stay in the intensive care unit. In these patients that survived, there was a significant decrease in albuminaemia and transferrin levels, no change in prealbuminaemia and a significant increase in ceruleoplasmin levels. Ten patients died during the study. They presented a greater decrease in albuminaemia and prealbuminaemia as compared with the survivors. There was no difference in the evolution of ceruleoplasmin and transferrin levels between the two groups. Thus, whilst the difference between survivors and non-survivors was significant for the group, for the individual patient the difference was too small to be of any prognostic value.

[Cerebral immunoblastic lymphoma and the acquired immunodeficiency syndrome]. / Lymphome immunoblastique cérébral et syndrome d'immunodéficit acquis.

A case of acquired immune deficiency syndrome (AIDS) is reported in a young homosexual Venezuelan male. It is of special interest to note the association of cytomegalovirus polyvisceral infection, frequent during AIDS, and a brain immunoblastic lymphoma, an infrequent tumour in that pathology.

Haemolytic-uraemic syndrome with bacteraemia caused by a new hybrid Escherichia coli pathotype.

We describe a new atypical Shiga-toxin-producing Escherichia coli (STEC) responsible for a severe episode of haemolytic-uraemic syndrome in an adult with a relapse associated with bacteraemia. This STECs train of serotype O80:H2 harboured stx2c and stx2d gene subtypes, the rare eae ξ variant and a ColV plasmid with a conserved virulence plasmidic region involved in virulence of human and avian extraintestinal pathogenic E. coli. This atypical hybrid pathotype, which represents a new threat, is a further demonstration that STEC may be a recipient for extraintestinal virulence factors and raises again the question of antibiotic therapy during STEC infection.

[Epidemiology of Streptococcus pyogenes invasive diseases in France (2007-2011)]. / Épidémiologie des infections invasives à Streptococcus pyogenes (France 2007-2011).

Group A Streptococcus (GAS) is a human pathogen responsible for a wide range of clinical manifestations. An increase of GAS invasive infections has been described since the mid 1980s. To study the French epidemiology of invasive infections (i) we characterized all GAS invasive strains received at the French National Reference Center for streptococci (CNR-Strep) between 2007 and 2011; (ii) we analyzed the epidemiological data on the corresponding strains. For each strain, emm genotype, superantigen genes and antibiotics susceptibility were determined. Among the 2 603 non redundant invasive GAS strains, 65.1 % (n=1 695) were isolated from blood culture. A streptococcal toxic shock syndrome (STSS) was described in 16.4 % (n=428) of cases, mostly associated with necrotizing fasciitis (NF), pleuropulmonary or osteoarticular infections (p ≤0.001). The case fatality rate was 10.6 %. A total of 102 different emm genotypes were identified. Three emm genotypes predominated, reaching nearly 60 % of the strains: emm 1 (26.7 %), emm 28 (16.4 %), and emm 89 (12.8 %). The proportion of each emm genotype varied according to the year and the age of patients. Among those < 15 years old, the three main genotypes were emm 1 (36.8 %), emm 12 (12.9 %) and emm 4 (9.5 %). The distribution of superantigen genes (SpeA, SpeC and Ssa) was restricted to several emm genotypes. Between 2007 and 2011, the rate of macrolides resistant GAS strains decreased from 7.8 to 5.5 %. emm 1 strains are still the most common especially in most severe clinical manifestations including STSS and NF.

Genotyping, local prevalence and international dissemination of ß-lactamase-producing Kingella kingae strains.

ß-lactamase production has been sporadically reported in the emerging Kingella kingae pathogen but the phenomenon has not been studied in-depth. We investigated the prevalence of ß-lactamase production among K. kingae isolates from different geographical origins and genetically characterized ß-lactamase-producing strains. Seven hundred and seventy-eight isolates from Iceland, the USA, France, Israel, Spain and Canada were screened for ß-lactamase production and, if positive, were characterized by PFGE and MLST genotyping, as well as rtxA, por, blaTEM and 16S rRNA sequencing. ß-lactamase was identified in invasive strains from Iceland (n=4/14, 28.6%), the USA (n=3/15, 20.0%) and Israel (n=2/190, 1.1%) and in carriage strains in the USA (n=5/17, 29.4%) and Israel (n=66/429, 15.4%). No French, Spanish or Canadian isolates were ß-lactamase producers. Among ß-lactamase producers, a perfect congruency between the different typing methods was observed. Surprisingly, all US and Icelandic ß-lactamase-producing isolates were almost indistinguishable, belonged to the major international invasive PFGE clone K/MLST ST-6, but differed from the four genetically unrelated Israeli ß-lactamase-producing clones. Representative strains of different genotypes produced the TEM-1 enzyme. K. kingae ß-lactamase producers exhibit a clear clonal distribution and have dissimilar invasive potential. The presence of the enzyme in isolates belonging to the major worldwide invasive clone K/ST-6 highlights the possible spread of ß-lactam resistance, and emphasizes the importance of routine testing of all K. kingae clinical isolates for ß-lactamase production.