Raffaella Marocco

In Vivo and In Vitro Effects of Antituberculosis Treatment on Mycobacterial Interferon-γ T Cell Response

Background In recent years, the impact of antituberculous treatment on interferon (IFN)-γ response to Mycobacterium tuberculosis antigens has been widely investigated, but the results have been controversial. The objective of the present study was: i) to evaluate longitudinal changes of IFN-γ response to M. tuberculosis-specific antigens in TB patients during antituberculous treatment by using the QuantiFERON-TB Gold (QFT-G) assay; ii) to compare the differences in T-cell response after a short or prolonged period of stimulation with mycobacterial antigens; iii) to assess the CD4+ and CD8+ T cells with effector/memory and central/memory phenotype; iv) to investigate the direct in vitro effects of antituberculous drugs on the secretion of IFN-γ. Principal Findings 38 TB patients was evaluated at baseline and at month 2 and 4 of treatment and at month 6 (treatment completion). 27 (71%) patients had a QFT-G reversion (positive to negative) at the end of therapy, while 11 (29%) TB patients remained QFT-G positive at the end of therapy. Among the 11 patients with persistent positive QFT-G results, six had a complete response to the treatment, while the remaining 5 patients did not have a resolution of the disease. All 27 patients who became QFT-G negative had a complete clinical and microbiological recovery of the TB disease. In these patients the release of IFN-γ is absent even after a prolonged 6-day incubation with both ESAT-6 and CFP-10 antigens and the percentage of effector/memory T-cells phenotype was markedly lower than subjects with persistent positive QFT-G results. The in vitro study showed that antituberculous drugs did not exert any inhibitory effect on IFN-γ production within the range of therapeutically achievable concentrations. Conclusions The present study suggests that the decrease in the M. tuberculosis-specific T cells responses following successful anti-TB therapy may have a clinical value as a supplemental tool for the monitoring of the efficacy of pharmacologic intervention for active TB. In addition, the antituberculous drugs do not have any direct down-regulatory effect on the specific IFN-γ response.

Severe and Persistent Depletion of Circulating Plasmacytoid Dendritic Cells in Patients with 2009 Pandemic H1N1 Infection

Background Dysregulation of host immune responses plays a critical role in the pathogenesis of severe 2009 pandemic H1N1 infection. Whether H1N1 virus could escape innate immune defense in vivo remains to be investigated. The aim of this study was to evaluate the pattern of innate immune response during human 2009 H1N1 infection. We performed the enumeration of circulating myeloid dendritic cells (mDC) and plasmacytoid DC (pDC) in blood from patients with H1N1 pneumonia shortly after the onset of symptoms and during follow-up at different intervals of time. The analysis of CD4 and CD8 count, CD38 T-cell activation marker and serum cytokine/chemokine plasma levels was also done. Methodology/Principal Findings Blood samples were collected from 13 hospitalized patients with confirmed H1N1-related pneumonia at time of admission and at weeks 1, 4, and 16 of follow-up. 13 healthy donors were enrolled as controls. In the acute phase of the disease, H1N1-infected patients exhibited a significant depletion in both circulating pDC and mDC in conjunction with a decrease of CD4 and CD8 T cell count. In addition, we found plasmatic hyperproduction of IP-10 and RANTES, whereas increase in T-cell immune activation was found at all time points. When we assessed the changes in DC count over time, we observed a progressive normalization of mDC number. On the contrary, H1N1-infected patients did not achieve a complete recovery of pDC count as values remained lower than healthy controls even after 16 weeks of follow-up. Conclusions H1N1 disease is associated with a profound depletion of DC subsets. The persistence of pDC deficit for several weeks after disease recovery could be due to H1N1 virus itself or to a preexisting impairment of innate immunity.

Antiviral treatment including entecavir plus tenofovir disoproxil fumarate for HBV reactivation following a rituximab-based regimen.

Entecavir and tenofovir disoproxil fumarate are potent and effective antiviral drugs that now represent recommended treatment options for chronic HBV infection. However, no or very limited clinical evidence is currently available on these drugs for the management of HBV reactivation in patients with haematological malignancies. Herein, we report a case of HBV reactivation in a patient with non-Hodgkins lymphoma following a rituximab-based regimen, and who was successfully treated with a combination antiviral treatment including entecavir and tenofovir disoproxil fumarate.

Multifunctional Analysis of CD4+ T-Cell Response as Immune-Based Model for Tuberculosis Detection

Mono- and multifunctional specific CD4+ and CD8+ T-cell responses were evaluated to improve the immune-based detection of active tuberculosis (TB) and latent infection (LTBI). We applied flow cytometry to investigate cytokines profile (IFN-γ, TNF-α, and IL-2) of T cells after stimulation with TB antigens in 28 TB-infected subjects (18 active TB and 10 LTBI) and 10 uninfected controls. Cytokines production by CD4+ T cells at single-cell levels was higher in TB-infected subjects than uninfected controls (P < 0.0001). Assigning to activated CD4+ T cells, producing any of the three cytokines, a cut-off >0.45%, it was possible to differentiate TB-infected (>0.45%) by uninfected subjects (<0.45%). Among TB-infected subjects, the frequencies of multifunctional CD4+ T cells, simultaneously producing all 3 cytokines, are lower in active TB than LTBI subjects (P = 0.003). Thus, assigning to triple-positive CD4+ T cells a cut-off <0.182%, TB-infected individuals could be classified as active TB subjects (<0.182%) or LTBI subjects (>0.182%). The magnitude of CD8+ T-cell responses showed no differences between active TB and LTBI. Multifunctional CD4+ T-cell responses could have the potential to identify at single time point subjects without TB infection and patients having active or latent TB.

Changes in inflammatory biomarkers in HCV-infected patients undergoing direct acting antiviral-containing regimens with or without interferon

Background and aims Increased levels of chemokine interferon-gamma (IFN-γ)-inducible protein-10 (CXCL10), soluble CD163 (sCD163) and soluble CD14 (sCD14) have been reported in HCV infection. The aim of this study was to compare, sCD163 and sCD14 levels in HCV-infected patients undergoing direct acting antiviral (DAA)-containing regimens with or without interferon (IFN). Methods sCD163, sCD14 and CXCL10 were longitudinally measured by ELISA in 159 plasma samples from 25 HCV-infected patients undergoing IFN-based treatment plus telaprevir or boceprevir and 28 HCV infected subjects treated with DAA IFN-free regimens. Twenty-five healthy donors (HD) were included as controls. Results At baseline CXCL10, sCD163 and sCD14 levels were higher in HCV-infected patients than in HD. CXCL10 and sCD163 levels were significantly decreased in responder (R) patients who achieved sustained virological response (SVR), with both IFN-based and IFN-free regimens, while they were persistently elevated in non-responders (NR) patients who stopped IFN-based treatments because of failure or adverse events. Conversely, sCD14 levels were apparently unchanged during therapy, but at the end of treatment the levels reached normal ranges. Comparing the two regimens, the extent of CXCL10 reduction was more pronounced in patients undergoing DAA IFN-free therapies, whereas sCD163 and sCD14 reduction was similar in the two groups. Interestingly, only in IFN-based regimens baseline sCD163 levels were significantly higher in NR than in R patients, while in the IFN-free treatment group also patients with high sCD163 plasma levels obtained SVR. At the end of therapy, even if the biomarkers were largely decreased, their levels remained significantly higher compared to HD. Only in the early fibrosis stages, sCD163 values tended to normalize. Conclusions These results indicate that IFN-free regimens including newer DAA induce an early and marked decrease in circulating inflammatory biomarkers. However, the full normalization of biomarkers was not obtained, especially in patients with advanced fibrosis, thus underlying the need for a treatment in the early stages of HCV infection.

Liver Fibrosis in HCV Monoinfected and HIV/HCV Coinfected Patients: Dysregulation of Matrix Metalloproteinases (MMPs) and Their Tissue Inhibitors TIMPs and Effect of HCV Protease Inhibitors

An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may contribute to liver fibrosis in patients with hepatitis C (HCV) infection. We measured the circulating levels of different MMPs and TIMPs in HCV monoinfected and HIV/HCV coinfected patients and evaluated the potential for anti-HCV therapy to modulate MMP and TIMP levels in HCV subjects. We analyzed 83 plasma samples from 16 HCV monoinfected patients undergoing dual or triple anti-HCV therapy, 15 HIV/HCV coinfected patients with undetectable HIV load, and 10 healthy donors (HD). Levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, TIMP-1, and TIMP-2 were measured by a SearchLight Multiplex Immunoassay Kit. MMP-2 and MMP-9 were the highest expressed MMPs among all the analyzed samples and their levels significantly increased in HCV monoinfected and HIV/HCV coinfected subjects compared to HD. TIMP-1 levels were significantly higher in HCV and HIV/HCV subjects compared to HD and were correlated with liver stiffness. These findings raise the possibility of using circulating TIMP-1 as a non-invasive marker of liver fibrosis in HCV infection. A longitudinal study demonstrated that MMP-9 levels significantly decreased (40% reduction from baseline) in patients receiving dual as well as triple direct-acting antivirals (DAA) anti-HCV therapy, which had no effect on MMP-2, TIMP-1, and TIMP-2. As the dysregulation of MMP-2 and MMP-9 may reflect inflammatory processes in the liver, the decrease of MMP-9 following HCV protease inhibitor treatment suggests a positive effect on the reduction of liver inflammation.

Postpartum fever in the presence of a fibroid: Sphingomonas paucimobilis sepsis associated with pyomyoma

BackgroundPyomyoma is a life-threatening complication of uterine leiomyoma. It may occur in post- menopausal women, during pregnancy and in the postpartum period. Fever may be the only manifestation during the early stages of the disease. We detail the first reported case of postpartum pyomyoma-related sepsis due to Sphingomonas paucimobilis, a Gram-negative bacillus that is gaining recognition as an important human pathogen.Case presentationA woman presented with an asymptomatic uterine fibroid and a two-week history of fever during the postpartum period. Suppurative uterine leiomyoma was diagnosed, and blood cultures grew Sphingomonas paucimobilis. The myoma was surgically removed from the uterus without hysterectomy. Intravenous antimicrobial therapy was given for fifteen days, and the patient was discharged from hospital in good condition.ConclusionPyomyoma should be considered in broad differential diagnosis of postpartum fever. This case highlights a unique disease manifestation of S. paucimobilis, an emerging opportunistic pathogen with increasing significance in the nosocomial setting.

Hepatitis B reactivation despite entecavir prophylaxis in a patient with chronic lymphocytic leukaemia receiving bendamustine

The qPCR and RFLP analyses disagreed in two cases (7.7%), where the rs8099917 genotype was determined by RFLP to be TT and qPCR indicated that it was TG. We arbitrarily considered the qPCR results to be the gold standard for analysis. The prevalence of IL28B CC (rs12979860) in European patients was 46% 7 and for TT (rs8099917) the prevalence was 51.5%, 9 whereas in the present study, the frequencies were 34.6% and 53.8%, respectively. Moreover, there was no statistical correlation between the TT IL28B (rs8099917) genotype and SVR, possibly due to the modest influence of this SNP and/ or the small sample size. To our knowledge, this is the first study evaluating the prevalence of IL28B SNPs in a Latin-American population. The analysis presented here was limited due to the low statistical power associated with the small sample size. Nevertheless, we observed a significant association between the IL28B CC genotype and SVR in Brazilian patients coinfected with HIV, as has been shown in case studies in Western Europe. References 1 Muir AJ, Bornstein JD, Killenberg PG et al. Peginterferon alfa-2b and ribavirin for the treatment of chronic hepatitis C in blacks and non-Hispanic whites. 3 Liu CH, Liu CJ, Lin CL et al. Pegylated interferon-a-2a plus ribavirin for treatment-naive Asian patients with hepatitis C virus genotype 1 infection: a multicenter, randomized controlled trial. et al. Genome-wide association of IL28B with response to pegylated interferon-a and ribavirin therapy for chronic hepatitis C. 7 Ralló n NI, Naggie S, Benito JM et al. Association of a single nucleotide polymorphism near the interleukin-28B gene with response to hepatitis C therapy in HIV/hepatitis C virus-coinfected patients. et al. IL28B SNP rs8099917 is strongly associated with pegylated interferon-a and ribavirin therapy treatment failure in HCV/HIV-1 coinfected patients. Sir, Several lines of evidence have shown that the occurrence of hepatitis B virus (HBV) reactivation is greatly reduced by the identification of high-risk patients and the use of prophylactic antiviral therapy. 1 Although the occurrence of HBV reactivation depends on HBV-DNA levels, the risk is also strongly linked to immunosuppressive drugs used as part of chemotherapeutic regimens. Bendamustine is a well-known chemotherapeutic agent with both alkylating and purine-like properties that has been recently used for the treatment of chronic lymphocytic leukaemia (CLL). Herein we report the case of a CLL patient with HBV infection [hepatitis B surface antigen (HBsAg) positive/HBV-DNA negative] who developed HBV reactivation during bendamustine …

Successful treatment of Stenotrophomonas maltophilia soft tissue infection with tigecycline: a case report.

Stenotrophomonas maltophilia is a nonfermentative, Gram-negative bacillus of increasing importance as a nosocomial pathogen. S. maltophilia has been implicated in a broad spectrum of clinical syndromes, including pneumonia, bloodstream infection, skin infections and surgical-site-related infections 1-4. S. maltophilia shares with other emerging multidrug-resistant Gram-negative organisms the ability to produce betalactamase enzymes involved in the hydrolysis of betalactam-based antibiotics, such as penicillins, cephalosporins and carbapenems. The treatment of nosocomial infections due to S. maltophilia is difficult and controversial 5. Trimethoprim-sulfamethoxazole (TMP-sMx) is the antimicrobial agent of choice, but it is bacteriostatic and increased resistance to this agent has been reported. in addition this pathogen is frequently associated with other multidrug resistant bacteria in the setting of polymicrobial infections. Tigecycline, a new semisynthetic glycylcycline, shows in vitro activity against S. maltophilia, but its efficacy in clinical settings is yet to be established 6. Here, we report a case of S. maltophilia surgical wound infection with surrounding soft tissue involvement which was successfully treated with tigecycline. A 17-year-old male was admitted to Emergency Department of our Hospital because of a compound fracture of his left tibia, rupture of femoral artery and wide soft tissues loss of the leg, due to a road accident. He underwent salvage combined surgery with reduction and osteosynthesis of fracture and arterial bypass. After 4 days, the patient developed fever (39.5°C), dehiscence of surgical incision with purulent drainage involving deep soft tissues of the leg. no evidence of muscle localization was found by computed tomography (CT) scan. Laboratory findings showed: hemoglobin 13.5 g/dl; white blood cells 12,500/mm3 with 80% of neutrophils; platelets, 170,000/mm3. Erythrocyte sedimentation rate was 52 mm/h (normal range, 0–30 mm/h), and his C-reactive protein concentration was 9.6 mg/dL (normal values 0.50 mg/dL). Liver and kidney function were normal. Empiric antibiotic treatment with piperacillintazobactam and teicoplanin was started; the patient also underwent hyperbaric oxygen therapy and surgical debridement of devitalized tissue from wound. Culture of the debridement material grew S. maltophilia susceptible to TMP-sMx (MiC<2 mg/mL) only. Thus, the patient started treatment with TMP-sMx (2 tablets of 160/800 mg 3 times daily), but after 4 days he developed an allergic rash. TMP-sMx was substituted with tigecycline (100 mg loading dose, then 50 mg every 12 hours). After 4 days of monotherapy with tigecycline, we observed a reduction in both fever and signs of local inflammation. This antibiotic was combined with cefepime (2 g i.v. twice daily) because of subsequent isolation of susceptible pseudomonas aeruginosa in the same site of infection. The combined antibiotic treatment was continued for 15 days and led to progressive resolution of local inflammation and microbiological eradication. S. maltophilia represents an emerging and difficult-to-treat nosocomial pathogen. The response to therapy is influenced by several factors, including the genotypic and phenotypic variability of S. maltophilia species, intrinsic resistance mechanisms, the ability to develop resistance during treatment and the lack of standardized susceptibility tests. in addition, in vitro findings cannot be translated into clinical practice because there are no randomized clinical trials which compare the efficacy of different antimicrobial regimens. The Clinical and Laboratory standards institute (CLsi) recommends the disk diffusion technique in order to establish the susceptibility of S. maltophilia to TMP-sMx, minocycline, and levofloxacin. Other agents may be used for therapy, but according to the CLsi, their performance has not been sufficiently studied to establish disk diffusion breakpoints 6. Tigecycline exhibits potent activity against a wide spectrum of bacteria, including methicillin-resistant Staphylococcus aureus, coagulase-negative staphylococci, penicillin-resistant Streptococcus pneumoniae, enterococcus faecium. Tigecycline has limited or no in vitro activity against p. aeruginosa, while the drug has been reported to be active in vitro against AcineREpRInt

Active HCV infection is associated with increased circulating levels of interferon-gamma (IFN-γ)-inducible protein-10 (IP-10), soluble CD163 and inflammatory monocytes regardless of liver fibrosis and HIV coinfection

BACKGROUND AND OBJECTIVE Interferon-gamma (IFN-γ)-inducible protein-10 (IP-10), soluble (s) CD163 and sCD14 play an important role in the pathogenesis of HCV and HIV infection and are involved in inflammation and liver fibrosis. The aim of the present study was to evaluate at a single time point, plasma soluble biomarkers and inflammatory monocytes subsets in different groups of subjects: (i) HIV monoinfected patients on suppressive ART; (ii) HIV/HCV coinfected patients on ART, with undetectable HIV viremia (including either subjects who had active HCV replication or those who cleared HCV); (iii) HCV monoinfected individual with active viral replication. METHODS Hundred and twenty-nine plasma samples were analyzed including HCV and HIV monoinfected patients, HIV/HCV coinfected patients, with active HCV infection (AHI) or with HCV viral clearance (VHC) and healthy donors (HD). Levels of IP-10, sCD163 and sCD14 were measured by ELISA. Absolute cell counts of monocyte subpopulations were enumerated in whole blood by using flow cytometric analyses. RESULTS IP-10 and sCD163 plasma levels were higher in HCV monoinfected and in AHI coinfected pts compared to HIV monoinfected and HD, whereas sCD14 levels were higher only in HIV monoinfected patients. Considering the degree of fibrosis, sCD163 and sCD14 levels positively correlated with kPa values (as assessed by fibroscan) and FIB-4 in HCV monoinfected group. On the other hand, IP-10 did not correlate with the fibrosis stage and it was found increased also in patients with low fibrosis. Moreover, we found an increase of the inflammatory NCM subset, in non-cirrhotic HCV subjects, while no alterations were observed in HIV, AHI and VHC. CONCLUSIONS Our study suggests a scenario in which active HCV infection is associated with a strong pro-inflammatory state, even in the initial stage of liver fibrosis, regardless the presence of HIV coinfection, thus underlying the need of an early anti-HCV treatment.