A case of surgical site infection with severe spondylitis caused by Streptococcus gordonii in an adult healthy woman: how to apply medico-legal method to deny malpractice hypothesis in suspected healthcare-associated infection.

We describe a rare case of severe primary spondylitis caused by Streptococcus gordonii in a 45-year-old immunocompetent woman with no relevant comorbidities. The surgical site infection arose after a L4-L5 microdiscectomy and resulted in severe clinical disability. Allegations of possible negligence as the cause prompted a forensic review to clarify the original source and transmission of this uncommon pathogen, which dismissed its cause as due to malpractice during treatment.

Hepatitis C virus infection: a challenge in the complex management of two cases of multidrug-resistant tuberculosis.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) requires lengthy use of second-line drugs, burdened by many side effects. Hepatitis C virus (HCV) chronic infection increases risk of drug-induced liver injury (DILI) in these patients. Data on MDR-TB patients with concurrent HCV chronic infection treated at the same time with second-line antitubercular drugs and new direct-acting antivirals (DAAs) are lacking. We evaluate if treating at the same time HCV infection and pulmonary MDR-TB is feasible and effective. CASES PRESENTATION: In this study, we described two cases of patients with pulmonary MDR-TB and concurrent HCV chronic infection cured with DAAs at a Tertiary Infectious Diseases Hospital in Italy. During antitubercular treatment, both patients experienced a DILI before treating HCV infection. After DAAs liver enzymes normalized and HCV RNA was undetectable. Then antitubercular regimen was started according to the institutional protocol, drawn up following WHO MDR-TB guidelines. It was completed without further liver side effects and patients were declared cured from both HCV infection and MDR-TB. CONCLUSIONS: We suggest to consider treatment of chronic hepatitis C with DAAs as a useful intervention for reintroduction of second-line antitubercular agents in those patients who developed DILI, reducing the risk of treatment interruption when re-exposed to these drugs.

First characterization of the CD4 and CD8 T-cell responses to QuantiFERON-TB Plus.

INTRODUCTION: QuantiFERON®-TB Gold Plus (QFT-Plus) is the new generation of QuantiFERON-TB Gold In-Tube test to identify latent tuberculosis infection (LTBI). QFT-Plus includes TB1 and TB2 tubes which contain selected Mycobacterium tuberculosis (Mtb) peptides designed to stimulate both CD4 and CD8 T-cells. Aim of this study is the flow cytometric characterization of the specific CD4 and CD8 T-cell responses to Mtb antigens contained within QFT-Plus. METHODS: We enrolled 27 active tuberculosis (TB) patients and 30 LTBI individuals. Following stimulation with TB1 and TB2, antigen-specific T-cells were characterized by flow cytometry. Data were also correlated with the grade of TB severity. RESULTS: TB1 mainly elicited a CD4 T-cell response while TB2 induced both CD4 and CD8 responses. Moreover, the TB2-specific CD4 response was detected for both active TB and LTBI patients, whereas the TB2-specific CD8 response was primarily associated with active TB (p = 0.01). CONCLUSIONS: To our knowledge, we report the first characterization of the CD4 and CD8 T-cell response to QFT-Plus. CD8 T-cell response is mainly due to TB2 stimulation which is largely associated to active TB. These results provide a better knowledge on the use of this assay.

Characterization of the CD4 and CD8 T-cell response in the QuantiFERON-TB Gold Plus kit.

OBJECTIVE/BACKGROUND: QuantiFERON-TB Gold In-Tube (QFT-GIT, Qiagen, Hilden, Germany) is an interferon-γ (IFN-γ) release assay designed to detect latent tuberculosis infection (LTBI). Although QFT-GIT has several advantages (mainly that it is not affected by the Bacille Calmette-Guérin vaccination), it has a poor sensitivity in immune-compromised individuals as it involves an immune response-based detection. Recently, QuantiFERON-TB Gold Plus (QFT-Plus) assay has been proposed as a new generation of QFT-GIT. QFT-Plus includes two tubes, TB1 and TB2 with Mycobacterium tuberculosis antigens to elicit a specific immune response. TB1 contains peptides derived from the antigens 6kDa early secretory antigenic target (ESAT-6) and 10kDa culture filtrate protein (CFP-10) (TB-7.7, present in QFT-GIT, has been removed), and it is designed to induce a specific CD4 T-cell response. TB2 contains newly designed peptides stimulating IFN-γ production by both CD4 and CD8 T cells. The additional peptides for eliciting CD8 T-cell responses have been included to increase the sensitivity of the test for LTBI detection. The aim of the study was to evaluate specific CD4 and CD8 T-cell responses to the M. tuberculosis antigens contained within the QFT-Plus test by flow cytometry in individuals with active TB and LTBI. METHODS: We enrolled 23 individuals with active TB and 30 individuals with LTBI. QFT-Plus assay and intracellular staining were performed. One million of peripheral blood mononuclear cells in 1ml of complete medium (RPMI 1640) were dispensed in QFT-Plus tubes. Following 16-24h stimulation, antigen-specific T cells were characterized by flow cytometry evaluating CD4, CD8, CD3 markers, and IFN-γ production. For statistical analysis, nonparametric tests were performed. RESULTS: We found that CD4 T-cell responses were induced by both TB1 and TB2. Differently, the CD8 T-cell response was mainly induced by TB2 and was significantly higher than that induced by TB1 (p=0.01). The frequency of Mtb specific T-cells observed in individuals with active TB was significantly higher than in those with LTBI (p=0.04). Finally, TB2-specific CD8 T-cell responses in individuals with active TB were associated with high radiological severity of lung lesions and microbiological diagnosis (based on M. tuberculosis isolation in sputum culture). CONCLUSION: This is the first characterization of CD4 and CD8 T-cell responses to QFT-Plus TB1 and TB2 tubes in individuals with active TB and LTBI enrolled in a low TB-endemic country such as Italy. We demonstrated that the increased sensitivity is a consequence of the ability of TB2 to induce a CD8 T-cell response which is mainly associated with active TB. This assay has the potential to be very useful in conditions of immune depression due to CD4 T-cell impairments.

Pandemic 2009 H1N1 virus infection associated with purpuric skin lesions: a case report.

INTRODUCTION: The influenza virus infection may be severe in non-immune people. Common complications of influenza virus include upper and lower respiratory tract infections, otitis media, myocarditis, acute respiratory distress syndrome and multi-organ failure. There have been cases of vasculitis following influenza vaccination, and rash and acute purpura may occur in certain viral infections. To the best of our knowledge, there are no reports concerning cases of systemic vasculitis associated with pandemic 2009 (H1N1) infection. CASE PRESENTATION: A 23-year-old Caucasian woman was hospitalized at the "L. Spallanzani" National Institute for Infectious Diseases in Rome, Italy. Clinical and radiological features including laboratory findings of this case are illustrated. Notably, the patient had fever, severe abdominal pain, hematuria, arthritis, and purpuric manifestations associated with a normal platelet count. Nasopharyngeal and rectal swabs revealed pandemic 2009 (H1N1) virus by reverse-transcriptase-polymerase-chain-reaction assay. Routine laboratory analyses showed elevated inflammatory parameters. The autoimmune panel tests were normal. Steroid therapy associated with oseltamivir achieved an evident and rapid improvement. On day seven the patient chose to leave the hospital against medical advice. CONCLUSION: Complications related to influenza infection can be life threatening, particularly in immunocompromised patients. Henoch-Schönlein purpura triggered by the novel influenza virus infection could be an attractive pathogenetic hypothesis. We have discussed both the diagnosis and the challenge of therapy protocols. Steroid therapy is part of the management of severe vasculitis. Our case suggests that steroid therapy associated with antivirals can prevent the risk of further complications such as hemorrhage and multi-organ failure during severe vasculitis, without enhancing the virulence of the influenza virus. The possible role of pandemic 2009 (H1N1) in the pathogenesis of hemorrhagic manifestations should be further investigated.

Determination of antituberculosis drug concentration in human plasma by MALDI-TOF/TOF.

Therapeutic drug monitoring allows to determine the best dosage regimen adapted to each patient optimizing the therapeutic benefits, while minimizing the risk for side effects. Here, the first methodological approach based on matrix-assisted laser desorption/ionization source equipped with tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry for the determination of the antituberculosis (anti-TB) drugs ethambutol, pyrazinamide, rifampicin, and streptomycin concentration in the plasma of tuberculosis-infected patients is reported. The volume of the plasma sample was 200 microL. Plasma samples were cleaned-up by protein precipitation and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with 200 microL of 50% methanol-0.03% formic acid solution (v/v), spiked with known amounts of anti-TB drugs, mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile-0.1% trifluoracetic acid solution; v/v), and spotted onto the MALDI-TOF/TOF sample target plate. The anti-TB drug concentration was determined by standard additions analysis. Regression of standard additions was linear over the whole anti-TB drug concentration range explored (the final anti-TB drug concentration ranged from 0.20 to 200 pmol/microL). The absolute recovery of the anti-TB drugs ranged between 87 and 110%. The minimal ethambutol, pyrazinamide, rifampicin, and streptomycin concentration detectable by MALDI-TOF/TOF is 0.08, 0.20, 0.12, and 0.15 pmol/microL, respectively.