Max Maurin

Comprehensive Diagnostic Strategy for Blood Culture-Negative Endocarditis: A Prospective Study of 819 New Cases

BACKGROUND. Blood culture-negative endocarditis (BCNE) may account for up to 31% of all cases of endocarditis. METHODS. We used a prospective, multimodal strategy incorporating serological, molecular, and histopathological assays to investigate specimens from 819 patients suspected of having BCNE. RESULTS. Diagnosis of endocarditis was first ruled out for 60 patients. Among 759 patients with BCNE, a causative microorganism was identified in 62.7%, and a noninfective etiology in 2.5%. Blood was the most useful specimen, providing a diagnosis for 47.7% of patients by serological analysis (mainly Q fever and Bartonella infections). Broad-range polymerase chain reaction (PCR) of blood and Bartonella-specific Western blot methods diagnosed 7 additional cases. PCR of valvular biopsies identified 109 more etiologies, mostly streptococci, Tropheryma whipplei, Bartonella species, and fungi. Primer extension enrichment reaction and autoimmunohistochemistry identified a microorganism in 5 additional patients. No virus or Chlamydia species were detected. A noninfective cause of endocarditis, particularly neoplasic or autoimmune disease, was determined by histological analysis or by searching for antinuclear antibodies in 19 (2.5%) of the patients. Our diagnostic strategy proved useful and sensitive for BCNE workup. CONCLUSIONS. We highlight the major role of zoonotic agents and the underestimated role of noninfective diseases in BCNE. We propose serological analysis for Coxiella burnetii and Bartonella species, detection of antinuclear antibodies and rheumatoid factor as first-line tests, followed by specific PCR assays for T. whipplei, Bartonella species, and fungi in blood. Broad-spectrum 16S and 18S ribosomal RNA PCR may be performed on valvular biopsies, when available.

Bartonella (Rochalimaea) quintana infections.

Bartonella (formerly Rochalimaea) quintana is the etiological agent of trench fever, a disease extensively reported during the World Wars. Recent molecular biology approaches have allowed dramatic extension of the spectrum of Bartonella infections. B. quintana is now also recognized as an etiological agent of fever and bacteremia, endocarditis, bacillary angiomatosis, and chronic lymphadenopathy. Human immunodeficiency virus-infected patients and/or homeless people are the most vulnerable to infection. Poverty and louse infestation were the main epidemiological factors associated with B. quintana infections during wartime. Although poverty and chronic alcoholism have been associated with modern cases of trench fever and bacteremia due to B. quintana in Europe and the United States, vectors for B. quintana have not been clearly identified and B. quintana has not been isolated from modern-day lice. Microscopic bacillary angiomatosis lesions are characterized by tumor-like capillary lobules, with proliferating endothelial cells. In vitro experiments have shown that B. quintana survives within endothelial cells and stimulates cell proliferation. These observations, together with the finding that lesions may regress when antibiotic therapy is administered, strongly suggest that B. quintana itself stimulates angiogenesis. Bartonella infections are characterized by a high frequency of relapses after brief courses of antibiotic therapy. It is to be noted that in vitro, although Bartonella species are highly susceptible to antibiotics, only the aminoglycosides have proved to be bactericidal. However, the most effective antibiotic regimen for Bartonella infections remains to be established.

MICs of 28 antibiotic compounds for 14 Bartonella (formerly Rochalimaea) isolates.

We assessed in vitro the antibiotic susceptibilities of 14 Bartonella isolates of the species B. quintana, B. vinsonii, B. henselae, and B. elizabethae. Columbia agar base supplemented with 5% horse blood was used as the antibiotic assay medium. Bacterial growth could be evaluated within 5 days after incubation of the plates at 37 degrees C in a 5% carbon dioxide atmosphere. The MICs at which 90% of isolates are inhibited (MIC90s) were 0.06 microgram/ml for penicillin G and amoxicillin and 0.25 microgram/ml for ticarcillin and cefotaxime. The MIC90s of oxacillin and cephalothin were 4 and 16 micrograms/ml, respectively. The MIC90s ranged from 1 to 4 micrograms/ml for aminoglycosides. Erythromycin, doxycycline, and rifampin displayed MIC90s of 0.12, 0.12, and 0.25 microgram/ml, respectively. MIC90s were 1 and 5 micrograms/ml for trimethoprim-and sulfamethoxazole, respectively, 64 micrograms/ml for fosfomycin, and 16 micrograms/ml for colistin and vancomycin. The study confirms the high levels of in vitro susceptibility of Bartonella agents to antibiotics.

From Q Fever to Coxiella burnetii Infection: a Paradigm Change

SUMMARY Coxiella burnetii is the agent of Q fever, or “query fever,” a zoonosis first described in Australia in 1937. Since this first description, knowledge about this pathogen and its associated infections has increased dramatically. We review here all the progress made over the last 20 years on this topic. C. burnetii is classically a strict intracellular, Gram-negative bacterium. However, a major step in the characterization of this pathogen was achieved by the establishment of its axenic culture. C. burnetii infects a wide range of animals, from arthropods to humans. The genetic determinants of virulence are now better known, thanks to the achievement of determining the genome sequences of several strains of this species and comparative genomic analyses. Q fever can be found worldwide, but the epidemiological features of this disease vary according to the geographic area considered, including situations where it is endemic or hyperendemic, and the occurrence of large epidemic outbreaks. In recent years, a major breakthrough in the understanding of the natural history of human infection with C. burnetii was the breaking of the old dichotomy between “acute” and “chronic” Q fever. The clinical presentation of C. burnetii infection depends on both the virulence of the infecting C. burnetii strain and specific risks factors in the infected patient. Moreover, no persistent infection can exist without a focus of infection. This paradigm change should allow better diagnosis and management of primary infection and long-term complications in patients with C. burnetii infection.

Human Tularemia in France, 2006–2010

BACKGROUND Tularemia is an endemic but rare disease in France. We describe the epidemiologic, clinical, diagnostic, treatment, and prognostic aspects of the disease in 101 consecutive patients investigated during a 5-year period (2006-2010). METHODS All tularemia cases confirmed at the French Reference Center for Tularemia (FRCT) were included. Data were collected both at the Institut de Veille Sanitaire (mandatory notification) and FRCT. Diagnostic methods included serological tests (microagglutination and indirect immunofluorescence assay), Francisella tularensis cultures, real-time polymerase chain reaction (RT-PCR) tests, and molecular identification of the F. tularensis subspecies involved. RESULTS The patient cohort consisted of 55 men and 46 women (sex ratio, 1.2; average age, 51.7 years), including 93 sporadic cases that occurred throughout France. Contaminations occurred predominantly through contact with or ingestion of lagomorphs (31.7%), tick bites (10.9%), or contaminated environments (7.9%). The glandular and ulceroglandular forms predominated (57.5% of cases), but 18.8% of patients experienced a systemic disease and 29.7% were hospitalized. Specific diagnosis was mainly based on serology, but 38.6% of patients had positive RT-PCR tests and 20.8% had a positive culture. F. tularensis subspecies holarctica was identified in 25 patients. All patients except 1 recovered from infection, but 38.6% experienced relapses despite appropriate antibiotic therapy. CONCLUSIONS The epidemiological and clinical aspects of tularemia in France are varied, suggesting different modes of contamination. The high rates of systemic diseases and hospitalization indicate that the more serious cases are more likely to be diagnosed and notified. RT-PCR tests may help to improve diagnosis and reporting of the disease.

Molecular Evaluation of Antibiotic Susceptibility: Tropheryma whipplei Paradigm

ABSTRACT Tropheryma whipplei, the agent of Whipples disease, grows fastidiously only in cell cultures without plaque production, and only three strains have been passaged. The formation of bacterial clumps in the supernatant precludes enumeration of viable bacteria and MIC determination. We evaluated the bacteriostatic effects of fluoroquinolones against two T. whipplei isolates by measuring the inhibition of the DNA copy number increase by real-time quantitative PCR. The analysis of the T. whipplei genome database allowed the identification not only of the gyrA gene but also the parC gene encoding the alpha subunit of the natural fluoroquinolone targets DNA gyrase (GyrA) and topoisomerase IV (ParC), respectively. The parC gene was detected in actinobacteria for the first time. High ciprofloxacin MICs (4 and 8 μg/ml) were correlated with the presence in T. whipplei GyrA and ParC sequences with an alanine residue at positions 83 and 80 (Escherichia coli numbering), respectively. Alanines at these positions have previously been associated with increased fluoroquinolone resistance in E. coli and mycobacteria. However, the MIC of levofloxacin was low (0.25 μg/ml). The same T. whipplei GyrA and ParC sequences were found in two other cultured strains and in nine uncultured tissue samples from Whipples disease patients, allowing one to speculate that T. whipplei is naturally relatively resistant to fluoroquinolones.

Evolution toward high-level fluoroquinolone resistance in Francisella species

OBJECTIVES Francisella tularensis, a CDC class A potential bioterrorism agent, is a Gram-negative bacterium responsible for tularaemia. Understanding the mechanisms of resistance to antibiotics used as first-line treatment is of major security relevance. METHODS We propagated the three parental reference strains Francisella tularensis subsp. holarctica live vaccine strain, Francisella novicida and Francisella philomiragia with increasing concentrations of ciprofloxacin, a fluoroquinolone used as curative and prophylactic treatment for tularaemia. This evolution procedure provided us with high-level ciprofloxacin-resistant mutants and all evolutionary intermediates towards high-level resistance. We determined the resistance levels to other fluoroquinolones (levofloxacin and moxifloxacin) and other antibiotic families (aminoglycosides, tetracyclines and macrolides) and characterized the genetic changes in the fluoroquinolone target genes encoding DNA gyrase and topoisomerase IV. RESULTS All high-level resistant mutants shared cross-resistance to the tested fluoroquinolones, while some also revealed striking levels of cross-resistance to other clinically relevant antibiotic classes. High-level resistant mutants carried one to three mutations, including some not previously reported. We mapped all mutations onto known topoisomerase three-dimensional structures. Along the pathways towards high-level resistance, we identified complex evolutionary trajectories including polymorphic states and additional resistance mechanisms likely to be associated with efflux processes. CONCLUSIONS Our data demonstrated the efficiency and speed of in vitro production of mutants highly resistant to fluoroquinolones in Francisella species. They emphasize the urgent need to identify all antibiotic resistance mechanisms in these species, develop molecular tools for their detection and design new therapeutic alternatives for tularaemia.

Tularaemia: clinical aspects in Europe.

Tularaemia is a zoonotic disease caused by Francisella tularensis, a Gram-negative, facultative intracellular bacterium. Typically, human and animal infections are caused by F tularensis subspecies tularensis (type A) strains mainly in Canada and USA, and F tularensis subspecies holarctica (type B) strains throughout the northern hemisphere, including Europe. In the past, the epidemiological, clinical, therapeutic, and prognostic aspects of tularaemia reported in the English medical literature were mainly those that had been reported in the USA, where the disease was first described. Tularaemia has markedly changed in the past decade, and a large number of studies have provided novel data for the disease characteristics in Europe. In this Review we aim to emphasise the specific and variable aspects of tularaemia in different European countries. In particular, two natural lifecycles of F tularensis have been described in this continent, although not fully characterised, which are associated with different modes of transmission, clinical features, and public health burdens of tularaemia.

Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila

OBJECTIVES Fluoroquinolone resistance has been poorly studied in Legionella pneumophila, an intracellular pathogen responsible for legionellosis. Our goal was to further characterize molecular mechanisms involved in fluoroquinolone resistance in this species. METHODS Eight independent lineages were founded from a common fluoroquinolone-susceptible L. pneumophila ancestor and propagated by serial passages in moxifloxacin-containing culture medium. We identified the substituted mutations that affected the DNA topoisomerase II-encoding genes, determined the order of substitution of the mutations leading to the stepwise MIC increases of moxifloxacin over evolutionary time and demonstrated their direct involvement in the resistance process. RESULTS Adaptation occurred through parallel stepwise increases in the moxifloxacin MICs up to 512-fold the MIC for the parental strain. Mutations affected the topoisomerase II-encoding genes gyrA, parC and gyrB, reflecting a high degree of genetic parallelism across the independent lineages. During evolution, the T83I change in GyrA occurred first, followed by G78D or S80R in ParC and D87N in GyrA, or S464Y or D426N in GyrB. By constructing isogenic strains, we showed that the progressive increase in resistance was linked to a precise order of mutation substitution, but also to the co-existence of several subpopulations of bacteria bearing different mutations. CONCLUSIONS Specific mutational trajectories were identified, strongly suggesting that intermolecular epistatic interactions between DNA topoisomerases underlie the mechanism of fluoroquinolone resistance in L. pneumophila. Our results suggest that L. pneumophila has strong potential to become resistant to fluoroquinolone compounds and warrant further investigation of resistance in clinical and environmental strains of this pathogen.

Can Whipple's Disease Be Transmitted by Gastroscopes?

OBJECTIVE To determine whether disinfection protocols currently used for gastroscopes are effective against cultures of Tropheryma whipplei. DESIGN The bactericidal activity of 2% glutaraldehyde and two peracetic acids on the Twist-Marseille strain of T. whipplei grown in cell monolayers was determined. PATIENTS Two patients who were diagnosed as having Whipples disease 3 years after they had had intestinal biopsies. RESULTS The disinfectants reduced bacteria by approximately 2 log to 3 log10 after 5 to 60 minutes of contact. CONCLUSION The bactericidal activity of a disinfectant is usually considered significant if it causes a 5 log10 or greater reduction in viable bacterial titers. Disinfecting gastroscopes with 2% glutaraldehyde or peracetic acids for 20 minutes may be insufficient to prevent transmission of T. whipplei on the instruments or stop false-positive results on polymerase chain reaction.