Frédérique Gouriet

Identification of Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT During the past 5 years, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.

The timing of surgery influences mortality and morbidity in adults with severe complicated infective endocarditis: a propensity analysis

AIMS To determine whether the timing of surgery could influence mortality and morbidity in adults with complicated infective endocarditis (IE). METHODS AND RESULTS In 291 consecutive adults with definite IE who underwent surgery during the active phase, we compared those operated on within the first week of antimicrobial therapy (n=95) to those operated on later (n=191). The impact of the timing of surgery on 6-month mortality, relapses, and postoperative valvular dysfunctions (PVD) was analysed using propensity score (PS) analyses. After stratification of the cohort into quintiles based on the PS, ≤1st week surgery was associated with a trend of decrease in 6-month mortality in the quintile of patients with the most likelihood of undergoing this early surgical management [quintile 5: 11% vs. 33%, odds ratio (OR)=0.18, 95% CI (confidence interval) 0.04-0.83, P=0.03]. Patients of this subgroup were younger, were more likely to have Staphylococcus aureus infections, congestive heart failure, and larger vegetations. Besides, ≤1st week surgery was associated with an increased number of relapses or PVD (16% vs. 4%, adjusted OR=2.9, 95% CI 0.99-8.40, P=0.05). CONCLUSION Surgery performed very early may improve survival in patients with the most severe complicated IE. However, a greater risk of relapses and PVD should be expected when surgery is performed very early.

Contribution of Systematic Serological Testing in Diagnosis of Infective Endocarditis

ABSTRACT Despite progress with diagnostic criteria, the type and timing of laboratory tests used to diagnose infective endocarditis (IE) have not been standardized. This is especially true with serological testing. Patients with suspected IE were evaluated by a standard diagnostic protocol. This protocol mandated an evaluation of the patients according to the modified Duke criteria and used a battery of laboratory investigations, including three sets of blood cultures and systematic serological testing for Coxiella burnetii, Bartonella spp., Aspergillus spp., Legionella pneumophila, and rheumatoid factor. In addition, cardiac valvular materials obtained at surgery were subjected to a comprehensive diagnostic evaluation, including PCR aimed at documenting the presence of fastidious organisms. The study included 1,998 suspected cases of IE seen over a 9-year period from April 1994 to December 2004 in Marseilles, France. They were evaluated prospectively. A total of 427 (21.4%) patients were diagnosed as having definite endocarditis. Possible endocarditis was diagnosed in 261 (13%) cases. The etiologic diagnosis was established in 397 (93%) cases by blood cultures, serological tests, and examination of the materials obtained from cardiac valves, respectively, in 348 (81.5%), 34 (8%), and 15 (3.5%) definite cases of IE. Concomitant infection with streptococci and C. burnetii was seen in two cases. The results of serological and rheumatoid factor evaluation reclassified 38 (8.9%) possible cases of IE as definite cases. Systematic serological testing improved the performance of the modified Duke criteria and was instrumental in establishing the etiologic diagnosis in 8% (34/427) cases of IE.

Dramatic Reduction in Infective Endocarditis-Related Mortality With a Management-Based Approach

BACKGROUND Despite improvements in medical and surgical therapy, infective endocarditis (IE) is still associated with a severe prognosis and remains a therapeutic challenge. We aimed to evaluate the impact of a standardized diagnostic and therapeutic protocol on mortality and to correlate the outcome with compliance with our management-based protocol. METHODS We conducted an observational before-after study that included 333 consecutive patients treated for IE at a referral center from 1991 to 2006, which was divided into 2 periods: period 1 (1991-2001), before implementation of our therapeutic protocol (n = 173), and period 2 (2002-2006), after implementation of our protocol (n = 160). Our protocol was created by a multidisciplinary task force including a sampling of biological specimens, the use of only 4 antimicrobial agents, a standardized duration of treatment, standardized surgical indications, and 1 year of close follow-up. Because our protocol was based on a local consensus by physicians and surgeons, it was not possible to randomize the study. RESULTS The 1-year mortality significantly decreased from 18.5% during period 1 to 8.2% during period 2 (hazard ratio, 0.41; 95% confidence interval, 0.21-0.79 [P = .008]). After multivariable analysis, the management during period 2 remained a strong protective factor (adjusted hazard ratio, 0.26; 95% confidence interval, 0.09-0.76 [P = .01]). During period 2, we observed a statistically significantly better compliance in antimicrobial therapy and fewer cases of renal failure. Deaths by embolic events and multiple organ failure syndrome also significantly decreased during period 2. CONCLUSION A dramatic reduction in mortality was observed during this study, suggesting that a management-based approach has a significant impact on IE outcome.

Evaluation of the LightCycler SeptiFast test in the rapid etiologic diagnostic of infectious endocarditis.

The SeptiFast test (Roche Diagnostics) is a new commercial molecular technique that has emerged for the detection of bacteria in blood. We compared in this study the sensitivity of blood culture to a commercially available broad-range real-time polymerase chain reaction (PCR) assay for the detection in blood of 19 bacterial species and six fungal species (SeptiFast test, Roche Diagnostics) in 63 patients with infectious endocarditis (IE). The SeptiFast test is not more sensitive for organisms such as Streptococci, Enterococci, and Staphylococcus aureus (11/29 versus 12/29 for blood culture). It has detected less commonly coagulase-negative Staphylococci (0/15 versus 3/15, P = 0.2) and significantly fewer other microorganisms (0/6 versus 4/6, P = 0.03). However, bacteria were detected from three IE treated by antibiotics, with blood culture negative on admission. The SeptiFast test may be useful in cases of IE in patients treated with antibiotics before admission.

First Case of Osteomyelitis Due to Shewanella algae

ABSTRACT Shewanella spp. are infrequently recovered from clinical specimens. We report here on the first case of osteomyelitis due to Shewanella algae. This bacterium, at first misidentified by phenotypic tests as Shewanella putrefaciens, was subsequently identified correctly as S. algae by 16S rRNA gene sequence analysis.

Detection by Immunofluorescence Assay of Bartonella henselae in Lymph Nodes from Patients with Cat Scratch Disease

ABSTRACT Laboratory diagnosis of Bartonella henselae infections can be accomplished by serology or PCR assay on biopsy samples. The purpose of our work was to assess immunofluorescence detection (IFD) in lymph node smears using a specific monoclonal antibody directed against B. henselae and a commercial serology assay (IFA) compared with PCR detection. Among 200 lymph nodes examined from immunocompetent patients, 54 were positive for B. henselae by PCR, of which 43 were also positive by IFD. Among the 146 PCR-negative lymph nodes, 11 were positive by IFD. Based on PCR results, the specificity of this new technique was 92.5%, the sensitivity was 79.6%, and the positive predictive value was 79.6%. At a cutoff titer of 64, the sensitivity of the IFA was 86.8% and the specificity was 74.1%. Diagnosis of cat scratch disease (CSD) may be improved, with a specificity of 100%, when the two tests (IFD and IFA) were negative; the sensitivity was 97.4% if one of the two tests was positive. Since PCR-based detection with biopsy samples is available only in reference laboratories, we suggest using IFD coupled with the commercial serology test for the diagnosis of CSD.

Outcome after surgical treatment performed within the first week of antimicrobial therapy during infective endocarditis: a prospective study.

BACKGROUND An increasing number of patients with infective endocarditis (IE) are operated on before the end of the first week of antimicrobial therapy. The mortality and morbidity of this specific group are unknown. AIMS To evaluate the outcome of patients with IE requiring cardiac surgery performed within the first week of antimicrobial therapy. METHODS All consecutive patients with a definite diagnosis of IE operated on within the first week of antimicrobial therapy were followed prospectively. Endpoints were in-hospital mortality and a combined endpoint of long-term cardiovascular death, recurrence and non-infective postoperative valvular dysfunction (PVD). The three main conditions requiring surgery, namely haemodynamic impairment, high embolic risk and periannular extension, were tested as potential predictors of outcome after adjustment for relevant variables. RESULTS Among the 95 patients included, surgery was performed a median time of 3 days after starting antimicrobial therapy. In-hospital mortality was 15%. The 3-year cumulative rates of the combined endpoint and of cardiovascular death were 38+/-7% and 27+/-7%, respectively. Recurrence occurred in 12% and PVD in 7%. Periannular extension was the main predictor of in-hospital death and the combined endpoint. CONCLUSION Despite the short time between starting antimicrobial therapy and performing surgery, the risk of death, recurrence and PVD does not appear excessively high. In the presence of periannular extension, however, surgery is associated with a greater risk of postoperative events.

Endocarditis Caused by Cardiobacterium valvarum

ABSTRACT A fastidious, gram-negative bacterium was isolated from the blood of a 51-year-old man who had acute infectious endocarditis (IE). Characterization of the organism through phenotypic and genotypic analyses revealed the causative role of Cardiobacterium valvarum. This is the third reported case of IE caused by C. valvarum.

Rickettsia slovaca Infection, France

To the Editor: Rickettsia slovaca was first isolated in 1968 in a Dermacentor marginatus tick collected in Slovakia, and serologic evidence of infection with this bacteria was reported in patients with enlarged lymph nodes and a scalp eschar after being bitten by a tick (1). However, the first proven case of R. slovaca infection was reported only in 1997 in France (2). This rickettsiosis is called tickborne lymphadenopathy (TIBOLA) because the most pronounced sign is lymph node enlargement. In Spain the same condition is called Dermacentor-borne-necrosis-erythema lymphadenopathy (3,4). In this study, we describe 14 new patients with TIBOLA from southern France who sought treatment from January 2004 to May 2005 and compare the features of these patients with those in whom Mediterranean spotted fever (MSF) was diagnosed during the same period. All the patients were referred to our center with a suspected rickettsial infection characterized by a tick bite located on the scalp, an inoculation eschar, and enlarged lymph nodes (Figure A1). For each patient, an acute-phase and a convalescent-phase serum sample were obtained for serologic analysis. Culture and polymerase chain reaction (PCR) were performed on tick, skin biopsy, or blood specimens. A multiple-antigen immunofluorescence assay (IFA) was performed by using 5 spotted fever group (SFG) rickettsial antigens: R. conorii conorii, R. slovaca, R. helvetica, R. sibirica mongolitimonae, and R. felis. Titers of at least 64 for immunoglobulin G (IgG) and 32 for IgM in acute-phase serum samples, evidence of seroconversion with 4-fold increases in IgG titers, or both, were considered as evidence of recent infections with a Rickettsia sp. (5). For serum specimens confirmed by IFA at the species level, Western immunoblotting and cross-adsorption assays procedures were performed as described elsewhere (6) by using R. conorii conorii and R. slovaca antigens. Patients with a definite serologic diagnosis at the species level were analyzed for their epidemiologic and clinical information. Culture from skin biopsy specimen and ticks were injected into human embryonic lung cells and cultivated into shell-vial culture as previously described (7). DNA was extracted from skin biopsy specimens, acute-phase serum samples, and ticks by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) (8). Standard PCR was performed with primers suitable for hybridization within the conserved region of genes coding for outer membrane protein A (ompA) and citrate synthase (gltA) (8). Among the 14 patients in a scalp lesion and cervical or occipital (1 case) lymph node enlargement developed after they were bitten by a tick, 9 were female (1 was pregnant) and 5 were male. The median (range) age was 34.9 (5-85) years with half of the patients <10 years of age. The incubation period ranged from 5 to 15 days (median 10.5 days; n = 7). Only 3 patients had fever. All patients fully recovered with doxycycline or, for the pregnant patient, josamycin therapy. Serology confirmed the diagnosis of R. slovaca infection for 10 patients by microimmunofluorescence and Western blot analysis after cross-adsorption studies (Table A1). R. slovaca was amplified by PCR for 7 cases, including 3 skin biopsy specimens, 3 Dermacentor marginatus ticks, and 1 acute-phase serum sample (Table A1). Three isolates (2 from skin biopsy specimens and 1 from a tick) were obtained by using the shell-vial culture assay. During the period of our study, in the same French region, 40 patients with MSF were clinically and laboratory diagnosed using the same procedures. The median (range) age was 54.2 (5–85) years with only 3 children <10 years of age (compared to 7/7 children with R. slovaca infection, p = 0.0015). MSF occurred mainly during the summer, whereas R. slovaca infection was seen during the colder months with 6 cases from October to January and 8 cases from February to May (Figure). In France, R. conorii has long been considered to be the only SFG rickettsiosis but R. slovaca may also be prevalent (9), contributing 25% of the cases in the present study. This organism is also a common cause of disease in Hungary and in La Rioja, Spain (3). These data suggest that TIBOLA mainly occurs in young children, affects women predominately, and occurs primarily during the colder months (9,10). As previously reported (9), we found that standard microimmunofluorescence serologic testing was insensitive and that Western blot is more useful and allows identification to the species level after cross-adsorption studies. Finally, DNA amplification by PCR from skin biopsy tissue, serum samples, or in ticks allowed confirmation of the diagnosis in only 50% of the cases, which suggests that other rickettsial species may be responsible for TIBOLA. Epidemiologic and clinical presentations are so characteristic that the clinical diagnosis should be considered in patients who have been bitten on the scalp during the colder months. In Europe, R. slovaca infection is likely to be a significant cause of cervical lymph node enlargement, and microbiologic investigation and tick analysis will underline the relative importance of this disease.