Hypervirulent Klebsiella Pneumoniae, an Emerging Cause of Endogenous Endophthalmitis in A French Center: A Comparative Cohort Study.

INTRODUCTION: Klebsiella pneumoniae (KP) is the most common cause of endogenous endophthalmitis (EE) in Asia, but data in Europe are scarce. We describe eight cases of KP EE compared to a cohort of EE in a French center. METHODS: EE cases were retrospectively studied between January 2014 and January 2021. KP EE cases were analyzed to assess clinical, microbiological features, and outcome. RESULTS: Among the 33 EE cases identified, the first causative agent (24%, n = 8) was KP, mainly (7/8) with hypervirulent phenotype (hvKP). All but one of these cases occurred from December 2019 to January 2021. Contrary to non-KP patients, KP patients had multiple extraocular infective foci (p = .006), all presented with liver abscesses (p < .001), 50% had cerebral involvement (p = .13). Visual outcome was poor in both groups. CONCLUSION: KP is an emerging cause of EE in a French center, consistently associated with liver abscesses, frequent cerebral involvement, and predominance of hvKP strains.

Diagnosis of Helicobacter pylori Infection in a Routine Testing Workflow: Effect of Bacterial Load and Virulence Factors.

Reliable diagnostic methods are mandatory for effective management of Helicobacter pylori infection. Histology and culture are the most common invasive methods in current practice, even if molecular methods are gaining in importance. The performance of these conventional methods varies significantly. We conducted a retrospective study of 1540 adults and 504 children with gastric biopsies taken during endoscopy to assess the impact of bacterial load and the cagA virulence factor on the performance of H. pylori infection testing. The association between virulence and histology findings was also investigated. With 23S rRNA qPCR confirmed by glmM amplification as the gold standard, culture and histology had lower sensitivity, 74.4% and 73.3%, respectively. However, their sensitivity was enhanced (>90%) in biopsies with high bacterial load (qPCR Ct < 30). Positive cagA status of the strain was associated with high bacterial load (94.9%), thus resulting in more frequent positive culture (94.3%) and H. pylori histology detection (91.7%) and more severe lesions on histology (p < 0.001). Conversely, the cagA status of the strains was negative in 110/119 (92.4%) of biopsies with low bacterial load (qPCR Ct < 30), 82/90 (91.1%) with negative H. pylori histology detection and 119/131 (90%) with negative culture findings (p < 0.001). This study highlights the low sensitivity of conventional culture and histology that may lead to false negative diagnosis if used alone. H. pylori quantification associated with cagA genotyping in routine workflow are essential for a sensitive and reliable diagnosis, to identify patients at high risk and to manage eradication therapies.

Characteristics of Helicobacter pylori strains isolated from Mauritanian patients.

BACKGROUND: Helicobacter pylori (H pylori) is responsible for various diseases including cancer It co-evolved with humans, and human migrations shaped the expansion and the diversity of strains around the world. The risk of developing a disease depends on virulence factors, mainly the cytotoxin-associated gene A protein (CagA). The aim of this study was to determine the cagA status in H pylori strains from Mauritanian patients and to search for a relationship with endoscopic and histologic findings. MATERIAL AND METHODS: H pylori was searched in gastric biopsies taken during endoscopy in patients with gastro-duodenal symptoms. RT-PCR was used for the diagnosis and resistance to clarithromycin. The cagA status was determined with PCR and the EPIYA-cagA polymorphism with sequencing. RESULTS: At all, 76/78 (97.4%) biopsies were positive. The rate of clarithromycin resistance was 4/76 (5.26%) due to the A2143G mutation, with a mixed population in 2 cases. The cagA gene was present in 23/76 (30.26%) biopsies, and the EPIYA motif was ABC in 21 (91.3%). High bacterial load and inflammation were significantly associated with cagA-positive status (P < .01). Phylogenetic analysis of the glmM and hspA genes highlighted a mixture of African and European genes in strains of H pylori isolated from patients of Moor origin. CONCLUSION: We report a high prevalence of H pylori infection in Mauritanian patients, a low rate of clarithromycin resistance (5.26%) and high bacterial load and inflammation associated with cagA-positive status. The phylogenetic analysis highlights the mix of different populations leading to the Moor ethnicity.

Molecular diagnosis of Helicobacter pylori infection in gastric biopsies: Evaluation of the Amplidiag® H. pylori + ClariR assay.

BACKGROUND: Adapted treatments for Helicobacter pylori infection, guided by determining antimicrobial resistance, are associated with high eradication rates. We evaluated the performance of the Amplidiag® H. pylori + ClariR PCR assay (Amplidiag® ) for detecting H. pylori and its clarithromycin resistance from gastric biopsies taken during endoscopy in comparison to culture and our "in-house" PCR. MATERIALS AND METHODS: A total of 127 gastric biopsies were analyzed (98 adults; 29 children). Culture, PCR Amplidiag® , and in-house PCR were performed in parallel. The in-house PCR combined amplification and sequencing of a 267-bp fragment of the H. pylori 23S rRNA gene. Discrepancies were controlled by amplification of glmM gene. RESULTS: For detection of H. pylori, Amplidiag® and the in-house PCR were concordant in 118 of 127 of cases: 66 negative and 52 positive. Discrepancies were observed in nine cases, all with low bacterial load: Amplidiag® did not detect seven biopsies positive on in-house PCR but detected two positive biopsies that were negative on in-house PCR. Among the 19 of 52 (36%) H. pylori cases resistant to clarithromycin, only four biopsies with mixed populations exhibited discordant results between the two PCR methods. The A2142T mutation was not detected by Amplidiag® . With the in-house PCR and amplified glmM gene as the reference method, the sensitivity and specificity of Amplidiag® was 88.5% (95% confidence interval 83-94.1) and 100%. CONCLUSION: This study demonstrated the high sensitivity of the PCR-based Amplidiag® H. pylori test, especially with low H. pylori load, and the probability of its clarithromycin resistance analysis. For clinical use, a well-designed trial with a large scale of samples may still be needed.

Comparison of the Diversilab® system with multi-locus sequence typing and pulsed-field gel electrophoresis for the characterization of Streptococcus agalactiae invasive strains.

Streptococcus agalactiae (or group B streptococcus; GBS) is a leading cause of neonatal morbidity and mortality in the developed countries. Several epidemiological typing tools have been developed for GBS to investigate the association between genotype and disease and to assess genetic variations within genogroups. This study compared the semi-automated repetitive sequence-based PCR Diversilab® system (DL) with MLST and pulsed field gel electrophoresis (PFGE) for determining the relatedness of invasive GBS strains. We analysed 179 unrelated GBS strains isolated from adult (n=108) and neonatal (n=71) invasive infections. By MLST, strains were resolved into 6 clonal complexes (CCs) including 23 sequence-types (STs), and 4 unique STs, whereas DL differentiated these isolates into 12 rep-PCR clusters (rPCs) and 9 unique rep-PCR types. The discriminatory power of both methods was similar, with Simpson's diversity indexes of 71.9% and 70.6%, respectively. However, their clustering concordance was low with Wallace concordance coefficients inferior to 0.4. PFGE was performed on 31 isolates representative of the most relevant DLrPCs clustered within the 3 major MLST CCs (CC-17, CC-23 and CC-1). As already observed with MLST, the concordance of DL with PFGE was low for all three CCs (Wallace coefficient <0.5), PFGE being more discriminative than rep-PCR. In summary, this work suggests that DL is less appropriate than MLST or PFGE to study GBS population genetic diversity.

Specific distribution within the Enterobacter cloacae complex of strains isolated from infected orthopedic implants.

Bacteria belonging to the Enterobacter genus are frequently isolated from clinical samples but are unusual causative agents of orthopedic implant infections. Twelve genetic clusters (clusters I to XII) and one sequence crowd (sequence crowd xiii) can be distinguished within the Enterobacter cloacae nomenspecies on the basis of hsp60 sequence analysis, and until now, none of these clusters could be specifically associated with a disease. In order to investigate if specific genetic clusters would be involved in infections of orthopedic material, two series of bacterial clinical isolates identified as E. cloacae by routine phenotypic identification methods were collected either from infected orthopedic implants (n = 21) or from randomly selected samples of diverse anatomical origins (control; n = 52). Analysis of the hsp60 gene showed that genetic clusters III, VI, and VIII were the most frequent genetic clusters detected in the control group, whereas cluster III was poorly represented among the orthopedic implant isolates (P = 0.006). On the other hand, E. hormaechei (clusters VI and VIII), but not cluster III, is predominantly associated with infections of orthopedic implants and, more specifically, with infected material in the hip (P = 0.019). These results support the hypothesis that, among the isolates within the E. cloacae complex, E. hormaechei and hsp60 gene sequencing-based cluster III are involved in pathogenesis in different ways and highlight the need for more accurate routine Enterobacter identification methods.

Invasive group B streptococcal infections in infants, France.

Clinical features and molecular characterization of 109 group B streptococci causing neonatal invasive infections were determined over an 18-month period in France. Sixty-four percent of the strains were from late-onset infections, and 75% were capsular type III. The hypervirulent clone ST-17 was recovered in 80% of meningitis cases.

Multiplex PCR assay for rapid and accurate capsular typing of group B streptococci.

We developed a simple, specific, and sensitive two-multiplex-PCR assay that enabled the detection of all known group B streptococcal (GBS) capsular polysaccharides. This test is well adapted for GBS capsular polysaccharide typing in large-scale epidemiological studies.

Rapid detection of the "highly virulent" group B Streptococcus ST-17 clone.

Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. Multilocus sequence typing (MLST) revealed that the sequence type ST-17 defines a "highly virulent" serotype III clone strongly associated with neonatal invasive infections. Our aim was to identify a target sequence enabling rapid, simple, and specific detection of this clone by a real-time PCR assay. Conventional methods for DNA manipulation and gene analyses were used to characterize the gbs2018 gene variant specific for ST-17 clone and to design ST-17- and GBS-specific primers. Conventional and real-time PCR assays were developed to detect GBS and ST-17 clones in bacterial cultures and directly on clinical samples. One hundred and fifty-six French GBS strains from various geographical areas in France isolated between 1990 and 2005 were screened by PCR with ST-17-specific primers. Forty strains were positive, and all were validated by MLST as ST-17. A representative sampling of 49 ST-17-PCR-negative strains was confirmed by MLST as non-ST-17. Real-time PCR was further used to directly test 85 vaginal samples. Among these, 13 were GBS-positive, and one was identified as ST-17. The association between strain invasiveness and ST-17 lineage in neonates with late onset disease was highly significant: 78% (P<0.0001) of strains isolated were ST-17. In conclusion, an ST-17-specific gbs2018 allele was identified and used to develop a sensitive and specific rapid-screening molecular assay for identifying ST-17 "highly virulent" GBS. Using this technique, accurate identification of women and neonates colonized by ST-17 can be readily achieved within less than 2 h.