Metagenomic Analysis of Microdissected Valvular Tissue for Etiological Diagnosis of Blood Culture-Negative Endocarditis.

BACKGROUND: Etiological diagnosis is a key to therapeutic adaptation and improved prognosis, particularly for infections such as endocarditis. In blood culture-negative endocarditis (BCNE), 22% of cases remain undiagnosed despite an updated comprehensive syndromic approach. This prompted us to develop a new diagnostic approach. METHODS: Eleven valves from 10 BCNE patients were analyzed using a method that combines human RNA bait-depletion with phi29 DNA polymerase-based multiple displacement amplification and shotgun DNA sequencing. An additional case in which a microbe was serendipitously visualized by immunofluorescence was analyzed using the same method, but after laser capture microdissection. RESULTS: Background DNA prevented any diagnosis in cases analyzed without microdissection because the majority of sequences were contaminants. Moraxella sequences were dramatically enriched in the stained microdissected region of the additional case. A consensus genome sequence of 2.4 Mbp covering more than 94% of the Moraxella osloensis KSH reference genome was reconstructed with 234X average coverage. Several antibiotic-resistance genes were observed. Etiological diagnosis was confirmed using Western blot and specific polymerase chain reaction with sequencing on a different valve sample. CONCLUSIONS: Microdissection could be a key to the metagenomic diagnosis of infectious diseases when a microbe is visualized but remains unidentified despite an updated optimal approach. Moraxella osloensis should be tested in blood culture-negative endocarditis.

Introduction to Measurement of Avidity of Anti-Coxiella burnetii IgG in Diagnosis of Q Fever.

Due to the atypical serological profile of some patients with primary Q fever infection who do not develop IgM against Coxiella burnetii, we developed an avidity test to distinguish recent or past infections. We tested 39 serum samples by immunofluorescence with conventional assay and after urea treatment from 26 patients at different stages of the disease. We observed a strong avidity in the 15 serum samples from patients with infections of >6 months and a low avidity for sera from patients with recent infections. A complete denaturation of the antibody-antigen complex was observed for patients for whom the time since the beginning of infection was <1 month and a mean of 2.06 ± 0.54 lowered titers when the infection was less than 3 months old. That was statistically significant compared to sera from patients with infections of greater than 6 months (mean 0.20 ± 0.41) and with infections between 3 and 6 months (mean, 1.17 ± 0.41) (P = 0.0022 and P < 0.0001, respectively). These results were visualized by Western blotting. We concluded that high avidity (≤1 lowered titer) ruled out infection during the last 6 months and that complete denaturation was related to an infection which had occurred within the previous 3 months. Between these two situations, the avidity test is inconclusive. We suggest using an avidity test for atypical Q fever serology that could be misclassified as residual antibodies (IgG against C. burnetii detected without active or recent infection) and for pregnant women risking obstetrical complications. This new test will dramatically improve the diagnosis and management of patients with Q fever.

Low antibodies titer and serological cross-reaction between Coxiella burnetii and Legionella pneumophila challenge the diagnosis of mediastinitis, an emerging Q fever clinical entity.

BACKGROUND: Coxiella burnetii is an intracellular and fastidious bacterium responsible of acute and persistent Q fever infection. Endocarditis and vascular infections are the most common serious complications of acute Q fever. CASE REPORT: We report the case of a 63-year-old man that presented a mediastinitis associated with a prosthetic vascular infection. Serological cross-reaction was observed between Coxiella burnetii, the agent of Q fever, and Legionella pneumophila with higher antibodies titer for L. pneumophila (IgG = 1:512) than for C. burnetii (phase I IgG = 1:400). We performed western blot with cross-adsorption that supports the diagnosis of C. burnetii infection. Two weeks later, a positive qPCR and culture for C. burnetii on swab taken from the mediastinal cutaneous fistula confirmed the definitive microbiological diagnosis of Q fever mediastinitis. CONCLUSION: Cross-reactivity between C. burnetii and Legionella spp. has long been known and should be considered in patients with persistent infections. It is important to establish the definite diagnosis because the antibiotic treatment regimens and duration are significantly different. To the best of our knowledge, we reported here the first case of mediastinitis associated to C. burnetii and we diagnosed this persistent infection despite low anti-C. burnetii phase I IgG levels.

A paradoxical Tropheryma whipplei western blot differentiates patients with whipple disease from asymptomatic carriers.

BACKGROUND: Tropheryma whipplei is a bacterium that causes Whipple disease. However, T. whipplei can be carried in the gut of asymptomatic people, which may lead to difficulty in the interpretation of positive stool sample test results. METHODS: This study included 60 patients with classic Whipple disease at the time of diagnosis and 26 T. whipplei carriers. Western blots testing for total immunoglobulin (Ig), IgG, IgM, and IgA were performed using glycosylated and deglycosylated T. whipplei. A blind test involving 10 patients and 10 carriers was performed. Sera samples from 32 treated patients were tested for total immunoglobulin. RESULTS: Total immunoglobulin from patients with classic Whipple disease exhibited either a lack of reaction (23 [38%] of 60 patients) or a decrease in reaction (33 [55%] of 60 patients) with a T. whipplei glycoprotein of 110 kDa after deglycosylation. Only 4 patients exhibited a stronger immune response than that which was observed for carriers (21 [81%] of 26 carriers). Five carriers presented a response profile similar to that for the patients. IgM (4 [7%] of 60 patients) or IgA (1 [2%] of 60 patients) responses were rarely observed but were exclusive to patients. Overall, results were consistent and reproducible. Antibiotic therapy had no effect on the serological profiles of the patients. CONCLUSIONS: Western blot serology is useful to distinguish between carriers and patients; paradoxical responses of the antibodies were investigated.

Western Immunoblotting for the Diagnosis of Enterococcus faecalis and Streptococcus gallolyticus Infective Endocarditis.

Blood culture-negative endocarditis (BCNE) remains a diagnostic challenge. In our center, despite a systematic and exhaustive microbiological diagnostics strategy, 22% of patients with BCNE remain without an identified etiology. In an effort to determine the relevance of using Western blot (WB) for the etiological diagnosis of BCNE in patients with early antibiotic use, we developed specific assays for the major infective endocarditis (IE) causative agents, namely, Staphylococcus aureus, Enterococcus faecalis, Streptococcus anginosus, and Streptococcus gallolyticus. Our technique was effective to identify the antigenic profiles of the four tested agents, but cross-reactions with S. aureus and S. anginosus antigens were frequent. A scoring method was developed for the diagnosis of E. faecalis and S. gallolyticus IE using the presence of reactivity to at least two antigenic bands for each bacterium and the positivity to at least one of the Ef300, Ef72, or Ef36 proteic bands for E. faecalis, and positivity for the two Sg75 and Sg97 proteic bands for S. gallolyticus. We tested these diagnostic criteria in a prospective cohort of 363 patients with suspected IE. Immunoblotting for the diagnosis of E. faecalis IE showed a sensitivity of 100% and a specificity of 99%. The positive and negative predictive values were 73 and 100%, respectively. Regarding S. gallolyticus infection, immunoblot had a sensitivity of 100% and a specificity of 95%. However, the positive predictive value was 22%, whereas the predictive negative value was 100%. Using WB, we identified a potential etiological agent in 4 of 14 BCNE cases with no identified pathogen. In conclusion, WB constitutes a promising and helpful method to diagnose E. faecalis or S. gallolyticus IE in patients with early antibiotic uptake and negative blood cultures.