Serial anti-tuberculous immune responses during the follow-up of patients with tuberculous pleurisy.

Little is known about the decay kinetics of interferon (IFN)-γ response and its influencing factors in tuberculous pleurisy. We enrolled thirty-two patients with tuberculous pleurisy prospectively and followed up at month 0, 6, and 9, at which time peripheral venous blood was drawn for interferon gamma release assay (IGRA) by means of QuantiFERON-TB Gold In-Tube (QFT-GIT). Demographic and clinical data were captured. To identify significant predictive factors influencing the IFN-γ response, multiple linear regression analyses were performed. Percentage of CD4+, CD8+, Vγ2Vδ2 T cells and Treg cells were measured by flow cytometry. The percentage of QFT-GIT-positive patients at baseline, month 6 and month 9 were 96.9% (30/32), 90.6% (29/32) and 84.4% (27/32), respectively. Quantitative IFN-γ response at baseline were significantly correlated with symptom duration (P = .003, R = 0.261) and age (P = .041, R = 0.132). Besides, the decreases of the IFN-γ response at month 6 and month 9 were positively correlated with the IFN-γ level at baseline. The dynamic tendency of the percentages of Treg cells was similar to the IFN-γ responses at each time-point. Quantitative IFN-γ response could be influenced by host immune status, instead of disease burden and anti-tuberculosis treatment. IGRA is probably not a useful biomarker of treatment efficacy in tuberculous pleurisy.

Cytokine profiles in cerebrospinal fluid of patients with meningitis at a tertiary general hospital in China.

BACKGROUND: There has been a great deal of evidence indicating that cytokines participate in meningeal inflammation. Different cytokine profiles may be presented in central nervous system (CNS) infection due to different pathogens. We have attempted to investigate cytokine profiles in cerebrospinal fluid (CSF) of patients with CNS infection. METHODS: Forty-three patients with CNS infection including tuberculous meningitis, purulent meningitis and cryptococcal meningitis were enrolled and 11 patients with normal CSF were enrolled as control group. The concentrations of Th1-, Th2- and Th17-type cytokines in CSF were detected using multiplex cytokine assay. Furthermore, the correlation between CSF cytokines and CSF parameters in CNS infection was analyzed. RESULTS: The CSF levels of IL-1ß, IL-4, IL-6, IL-10, IL-17, IL-23, IL-33, IFN-γ, TNF-α and sCD40L among the patients with CNS infection were all higher than control group (all P < 0.05). A remarkable elevation of CSF IL-6 in the patients with CNS infection was observed with the least overlap of the CSF concentrations compared to controls. Moreover, CSF IL-6 levels were strongly negatively correlated with CSF glucose and the CSF/blood glucose ratio (r = -0.4375, P = 0.0042; r = -0.4991, P = 0.0009). CONCLUSIONS: The excessive activation of immune response characterized by elevated levels of CSF Th1-, Th2- and Th17-type cytokines has been observed during CNS infection. Furthermore, we observed negative correlations between CSF IL-6 levels and CSF glucose and CSF/blood glucose ratio in CNS infection. And we suggested that combined CSF IL-6 levels with CSF glucose may serve as a novel biomarker pool for the differential of CNS infection.

Rapid detection of respiratory organisms with FilmArray respiratory panel and its impact on clinical decisions in Shanghai, China, 2016-2018.

BACKGROUND: In this study, we evaluated the diagnostic potential and clinical impact of an automated multiplex PCR platform (the FilmArray Respiratory Panel; FA-RP), specially designed for pathogen detection in respiratory tract infections in adults with unexplained pneumonia (UP). METHODS: A total of 112 UP patients in Shanghai, China, were enrolled prospectively and assessed using the FA-RP from October 2016 to March 2018. We examined the test results and their influence on clinical decisions. Furthermore, as a control group, we retrospectively obtained the clinical data of 70 UP patients between October 2014 and March 2016 (before the FA-RP was available). The two patient groups were compared with respect to factors, including general antimicrobial use and defined daily dose (DDD) numbers. RESULTS: Between October 2016 and March 2018, the positive rate obtained using FA-RP for UP was 76.8%. The primary pathogens in adults with UP were Influenza A/B (47.3%, 53/112). Compared with the patients before FA-RP was available, patients who underwent FA-RP testing had higher rates of antiviral drug use and antibiotic de-escalation during clinical treatment. FA-RP significantly decreased the total DDDs of antibiotic or antifungal drugs DDDs by 7 days after admission (10.6 ± 2.5 vs 14.1 ± 8.8, P < .01). CONCLUSIONS: The FA-RP is a rapid and sensitive nucleic acid amplification test method for UP diagnosis in adults. The application of FA-RP may lead to a more accurately targeted antimicrobial treatment and reduced use of antibiotic/antifungal drugs.

Late prosthetic valve endocarditis with Mycobacterium tuberculosis after the Bentall procedure.

BACKGROUND: Prosthetic valve endocarditis (PVE) is a rare but severe complication of valve replacement surgery, with an incidence rate of 0.3-1.2% per patient-year. At present, staphylococci are the predominant causative microorganism of PVE. Herein, we report a confirmed case of late PVE in a mechanical aortic valve caused by Mycobacterium tuberculosis. CASE PRESENTATION: A 32-year-old immunocompetent man with recurrent fever and 5-kg weight loss had a history of having undergone the Bentall procedure due to congenital heart disease. Nine years after the operation, he developed a paravalvular abscess in the mechanical aortic valve, presented with evidence of pulmonary tuberculosis on CT scan and was diagnosed with tuberculous endocarditis. This case report highlights a rare and non-negligible example of tuberculous endocarditis involving a mechanical valve. CONCLUSIONS: Tuberculous PVE should be considered in patients with a history of valve replacement, recurrent fever, unexplained weight loss, pulmonary tuberculosis and meaningful valvular findings on echocardiogram.

Genetic influence of Toll-like receptors on non-HIV cryptococcal meningitis: An observational cohort study.

BACKGROUND: Cryptococcal meningitis (CM) is a significant source of mortality, the pathogenesis of which has not been fully understood, especially in non-HIV infected populations. We aimed to explore the potential genetic influence of Toll-like receptor (TLR) on non-HIV CM. METHODS: This observational cohort study was done in two stages: a discovery stage and a validation stage. A case-control genetic association study was conducted between 159 non-HIV CM patients and 468 healthy controls. TLR SNPs significantly related to susceptibility went further validation in a second cohort of 583 subjects from a certain district. Associations among TLR SNPs, cerebrospinal fluid (CSF) cytokine concentrations, and clinical severity were explored in a third cohort of 99 previously untreated non-HIV CM patients. Logistic regression model was used to determine the independent predictors for disease severity. FINDINGS: In the discovery stage, eight TLR SNPs exhibited significant genetic susceptibility to non-HIV CM, one of which was validated in a population validation of HIV-infected cases while none survived in non-HIV cases. CSF cytokine detections showed that 18 cytokines were significantly over-expressed in severely ill patients. Two of the 8 SNPs (rs5743604 and rs3804099) were also significantly associated with disease severity. Specifically, the rs3804099 C/T genotype was further found to be correlated to 12 of the 18 up-regulated cytokines in severe patients. In addition, high levels of interleukin (IL)-10 in CSF (OR 2·97, 95% CI 1·49-5·90; p = 0·002) was suggested as an independent predictor for severity after adjusted for possible confounders. INTERPRETATION: TLR participates in both the occurrence and the pathogenesis of non-HIV CM. The in situ immune responses of CM were under genetic influence of TLR and contributed to disease severity. FUND: National Natural Science Foundation of China and National Key Basic Research Program of China (973 Program).

The expressions and roles of different forms of IL-22 in Mycobacterium tuberculosis infection.

Despite evidence suggesting an anti-Mycobacterium tuberculosis effector function of CD4+ T cells that produce and retain IL-22 in macaques, the general role of IL-22 in tuberculosis infection is still poorly characterized. To explore the immune mechanism in the pathogenesis of tuberculosis in humans, here we evaluated different forms of IL-22 in populations with different tuberculosis infection statuses. We enrolled 156 subjects including 49 patients with pulmonary tuberculosis, 27 patients with tuberculous pleurisy (TPE), 38 individuals with latent tuberculous infection (LTBI) and 42 healthy controls (HC). We found significantly higher IL-22 levels at the tuberculosis infection site than in the peripheral blood as well as higher antigen-specific IL-22 levels in the culture supernatant for patients with active tuberculosis than in healthy controls. The proportions of IL-22 + CD4+ T and IL-22 + CD8+ T cells in patients with active tuberculosis were significantly higher than those in the latent tuberculosis infection group and the healthy control group, based on intracellular cytokine staining. However, surprisingly, we found membrane-bound IL-22+ T cells, including CD4+ T cells and CD8+ T cells, by surface staining, especially in patients with active tuberculosis. Furthermore, the expression of membrane-bound IL-22 significantly decreased after drug therapy. In conclusion, our results suggest that IL-22 has various roles in tuberculosis immune responses. In particular, membrane-bound IL-22+ T cells may play important roles in the human immune response to Mycobacterium.

A combination of the QuantiFERON-TB Gold In-Tube assay and the detection of adenosine deaminase improves the diagnosis of tuberculous pleural effusion.

The differential diagnosis of tuberculous pleural effusion (TPE) and malignant pleural effusion (MPE) remains difficult despite the availability of numerous diagnostic tools. The current study aimed to evaluate the performance of the whole blood QuantiFERON-TB Gold In-Tube (QFT-GIT) assay and conventional laboratory biomarkers in differential diagnosis of TPE and MPE in high tuberculosis prevalence areas. A total of 117 patients with pleural effusions were recruited, including 91 with TPE and 26 with MPE. All of the patients were tested with QFT-GIT, and the conventional biomarkers in both blood and pleural effusion were detected. The level of antigen-stimulated QFT-GIT in the whole blood of TPE patients was significantly higher than that of MPE (2.89 vs 0.33 IU/mL, P<0.0001). The sensitivity and specificity of QFT-GIT for the diagnosis of TPE were 93.0% and 60.0%, respectively. Among the biomarkers in blood and pleural effusion, pleural adenosine deaminase (ADA) was the most prominent biomarker, with a cutoff value of 15.35 IU/L. The sensitivity and specificity for the diagnosis of TPE were 93.4% and 96.2%, respectively. The diagnostic classification tree from the combination of these two biomarkers was 97.8% sensitive and 92.3% specific. Ultimately, the combination of whole blood QFT-GIT with pleural ADA improved both the specificity and positive predictive value to 100%. Thus, QFT-GIT is not superior to pleural ADA in the differential diagnosis of TPE and MPE. Combined whole blood QFT-GIT and pleural ADA detection can improve the diagnosis of TPE.

Treating chronic hepatitis B virus: Chinese physicians' awareness of the 2010 guidelines.

AIM: To investigate Chinese physicians' awareness of the 2010 guidelines on the treatment of chronic hepatitis B virus (HBV) infection. METHODS: This was a quantitative survey that investigated the characteristics and practices of physicians who were treating patients with hepatitis B, the profile of their patients and physician practices regarding the diagnosis and treatment of HBV at the time of the survey. Participants were randomly selected from available databases of Chinese physicians and requested to complete either an online or paper-based survey. Data from the survey responses were analysed. For data validation and interpretation, qualitative indepth interviews were conducted with 39 of the respondents. RESULTS: Five-hundred completed surveys, from 663 physicians were available for analysis. A mean of 175 chronic hepatitis B (CHB) patients was seen by each physician every month, of whom 85 (49%) were treated in line with therapeutic indications stated in the 2010 guidelines. A total of 444 (89%) physicians often (> 60% of the time) adhered to the guidelines. Most physicians used antiviral medications as recommended. For patients with compensated and decompensated cirrhosis, 342 (68%) and 336 (67%) of physicians, respectively, often followed the recommendation to use potent nucleos(t)ide analogues with a high genetic barrier to resistance, using the appropriate treatment more than 60% of the time. Physicians from infectious disease or liver disease departments were better informed than those from gastrointestinal or other departments. CONCLUSION: The majority of Chinese physicians often adhere to Chinese 2010 CHB guidelines and are well-informed about the use of antiviral medications for hepatitis B.

Hierarchy Low CD4+/CD8+ T-Cell Counts and IFN-γ Responses in HIV-1+ Individuals Correlate with Active TB and/or M.tb Co-Infection.

OBJECTIVE: Detailed studies of correlation between HIV-M.tb co-infection and hierarchy declines of CD8+/CD4+ T-cell counts and IFN-γ responses have not been done. We conducted case-control studies to address this issue. METHODS: 164 HIV-1-infected individuals comprised of HIV-1+ATB, HIV-1+LTB and HIV-1+TB- groups were evaluated. Immune phenotyping and complete blood count (CBC) were employed to measure CD4+ and CD8+ T-cell counts; T.SPOT.TB and intracellular cytokine staining (ICS) were utilized to detect ESAT6, CFP10 or PPD-specific IFN-γ responses. RESULTS: There were significant differences in median CD4+ T-cell counts between HIV-1+ATB (164/µL), HIV-1+LTB (447/µL) and HIV-1+TB- (329/µL) groups. Hierarchy low CD4+ T-cell counts (<200/µL, 200-500/µL, >500/µL) were correlated significantly with active TB but not M.tb co-infection. Interestingly, hierarchy low CD8+ T-cell counts were not only associated significantly with active TB but also with M.tb co-infection (P<0.001). Immunologically, HIV-1+ATB group showed significantly lower numbers of ESAT-6-/CFP-10-specific IFN-γ+ T cells than HIV-1+LTB group. Consistently, PPD-specific IFN-γ+CD4+/CD8+ T effector cells in HIV-1+ATB group were significantly lower than those in HIV-1+LTB group (P<0.001). CONCLUSIONS: Hierarchy low CD8+ T-cell counts and effector function in HIV-1-infected individuals are correlated with both M.tb co-infection and active TB. Hierarchy low CD4+ T-cell counts and Th1 effector function in HIV-1+ individuals are associated with increased frequencies of active TB, but not M.tb co-infection.

Potential diagnostic value of serum/pleural fluid IL-31 levels for tuberculous pleural effusion.

The aim of this study was to explore the diagnostic value of IL-31 levels in the pleural fluid and plasma to differentially diagnose tuberculous and malignant pleural effusion. We enrolled 91 cases, including tuberculous pleural effusion (TPE, n = 50), malignant pleural effusion (MPE, n = 41), other cases including pneumonia with pleural fluid, pulmonary tuberculosis and healthy people as controls. Whole blood was stimulated with the M. tuberculosis-specific antigens and plasma was collected. The multiplex bead-based cytokine immunoassay was employed to measure the levels of various cytokines. IL-31 was found to be the most prominent cytokine (P < 0.0001), and with an optimal cut-off value of 67.5 pg/mL, the sensitivity and specificity for the diagnosis of TPE were 86% and 100%, respectively. Furthermore, the tuberculosis-specific IL-31 levels in the plasma of TPE patients were higher than that of MPE patients (P = 0.0002). At an optimal cut-off value of 23.9 pg/mL, the sensitivity and specificity for the diagnosis of TPE were 92.9% and 85.7%, respectively. Ultimately, the combination of pleural fluid with the plasma tuberculosis-specific IL-31 levels improved the sensitivity and specificity to 94.0% and 95.1%, respectively. Thus, we identified a novel biomarker for the diagnosis of TPE for clinical application.

Characterization of CD4/CD8+ αß and Vγ2Vδ2+ T cells in HIV-negative individuals with different Mycobacterium tuberculosis infection statuses.

BACKGROUND: The immune responses of T cell subsets among patients with different Mycobacterium tuberculosis (M.tb) infection statuses [i.e., active tuberculosis (ATB), latent tuberculosis infection (LTBI) and non-infection (healthy control, HC)] have not been fully elucidated in HIV-negative individuals. Specifically, data are limiting in high tuberculosis epidemic regions in China. To investigate the distributions and functions of T cell subsets (i.e., CD3+, CD4+, CD8+ αß and Vγ2Vδ2+ T cells) in HIV-negative subjects with different M.tb infection statuses, we conducted a case-control study that enrolled 125 participants, including ATB patients (n = 46), LTBI subjects (n = 34), and HC (n = 45). RESULTS: An IFN-γ release assay (IGRA) was employed to screen LTBI subjects. Whole blood cell surface staining and flow cytometry were used to detect phenotypic distributions of T cells in the peripheral blood mononuclear cells (PBMCs) and tuberculous pleural fluid mononuclear cells (PFMCs). PPD and the phosphorylated antigen HMBPP were employed as stimulators for the detection of M.tb antigen-specific T cell functions via intracellular cytokine staining (ICS). The absolute numbers of T cell subsets, including CD3+ CD4+, CD3+ CD8+ αß and Vγ2Vδ2+ T cells, were significantly reduced in active tuberculosis compared with latent tuberculosis or the healthy controls. Importantly, PPD-specific CD3+ CD4+ and CD3+ CD8+ αß T cells and HMBPP-specific Vγ2Vδ2+ T cells in ATB patients were also significantly reduced compared to the LTBI/HC subjects (P<0.05). In contrast, the proportion of CD4+ T cells in PFMCs was higher compared to PBMCs, while CD8+ and Vγ2Vδ2+ T cells in PFMCs were lower compared to PBMCs (all P < 0.05). PPD-specific CD4+ T cells predominated among CD3+ T cells in PFMCs. CONCLUSIONS: Cellular immune responses are impaired in ATB patients. Antigen-specific CD4+ T cell may migrate from the periphery to the lesion site, where they exert anti-tuberculosis functions.

Histological outcome for chronic hepatitis B patients treated with entecavir vs lamivudine-based therapy.

AIM: To compare the histological outcome of chronic hepatitis B (CHB) patients treated with entecavir (ETV) or lamivudine (LAM)-based therapy. METHODS: We conducted a retrospective analysis of data from 42 CHB patients with advanced fibrosis (baseline Ishak score ≥ 2) or cirrhosis who were treated with ETV or LAM-based therapy in Beilun People's Hospital, Ningbo between January 2005 and May 2012. The patients enrolled were more than 16 years of age and underwent a minimum of 12 mo of antiviral therapy. We collected data on the baseline characteristics of each patient and obtained paired liver biopsies pre- and post-treatment. The Knodell scoring system and Ishak fibrosis scores were used to evaluate each example. An improvement or worsening of necroinflammation was defined as ≥ 2-point change in the Knodell inflammatory score. The progression or regression of fibrosis was defined as ≥ 1-point change in the Ishak fibrosis score. The continuous variables were compared using t-test or Mann-Whitney test, and the binary variables were compared using χ(2) test or Fisher's exact test. The results of paired liver biopsies were compared with a Wilcoxon signed rank test. RESULTS: Nineteen patients were treated with ETV and 23 patients were treated with LAM therapy for a mean duration of 39 and 42 mo, respectively. After long-term antiviral treatment, 94.74% (18/19) of the patients in the ETV arm and 95.65% (22/23) in the LAM arm achieved an HBV DNA level less than 1000 IU/mL. The majority of the patients (94.74% in the ETV arm and 73.91% in the LAM arm) had normalized ALT levels. The median Knodell necroinflammatory score decreased from 11 to 0 in the patients receiving ETV, and the median Knodell score decreased from 9 to 3 in the patients receiving LAM (P = 0.0002 and < 0.0001, respectively). The median Ishak fibrosis score showed a 1-point reduction in ETV-treated patients and a 2-point reduction in LAM-treated patients (P = 0.0019 and 0.0205, respectively). The patients receiving ETV showed a more significant improvement in necroinflammation than the LAM-treated patients (P = 0.0003). However, there was no significant difference in fibrotic improvement between the two arms. Furthermore, two patients in each arm achieved a fibrosis score of 0 post-treatment, which indicates a full reversion of fibrosis after antiviral therapy. CONCLUSION: CHB patients with advanced fibrosis or cirrhosis benefit from antiviral treatment. ETV is superior to LAM therapy in improving necroinflammatory but not fibrotic outcome.

Epidemiology and the prognosis of healthcare-associated infective endocarditis in China: the significance of non-nosocomial acquisition.

Limited research has been conducted on healthcare-associated infective endocarditis (HAIE), although it is of increasing importance. The aim of this study is to compare the epidemiology, clinical characteristics, and prognosis of community-acquired IE (CA-IE) with HAIE and non-nosocomial healthcare-associated IE (NNHCA-IE). A retrospective, consecutive case-series analysis was organized and performed during the 20-year study period in Huashan Hospital, Shanghai, China. A total of 154 patients were enrolled, including 126 (81.8%) who had CA-IE and 28 (18.2%) who had HAIE, among whom 20 (71.4%) had non-nosocomial IE. Patients with HAIE compared to patients with CA-IE had poorer clinical conditions (Charlson comorbidity index ≥2: 35.7% vs. 15.1%, P = 0.012; immunosuppressive therapy: 21.4% vs. 4.0%, P = 0.005), underwent more prosthetic valve replacement (35.7% vs. 7.1%, P <0.001), had less streptococcus infection (16.7% vs. 51.1%, P = 0.007) but more atypical bacterial infection (50.0% vs. 21.1%, P = 0.017) and poorer outcomes (17.9% vs. 4.0%, P = 0.019). It is noteworthy that the results were quite similar between the comparison of patients with NNHCA-IE and those with CA-IE. Overall, in-hospital mortality was 6.5%. The IE acquisition site and low serum albumin levels (odds ratio (OR): 0.8; P = 0.04) were significantly associated with an increased risk of mortality. Nosocomial IE patients had an 8.3-fold and NNHCA-IE patients had 6.5-fold increase in the risk of mortality compared to CA-IE patients. In conclusion, HAIE and NNHCA-IE have important epidemiological and prognostic implications. Because NNHCA-IE usually occurs in patients residing in the community, it is suggested that these patients should be identified and treated by the community primary care clinical staff as early as possible.

Dectin-2 polymorphism associated with pulmonary cryptococcosis in HIV-uninfected Chinese patients.

Dectin-2 is a C-type lectin receptor that can recognize critical structures of fungi and involve in the host immune response after pulmonary fungal infections. We aimed to investigate the association between Dectin-2 genetic polymorphisms and cryptococcosis among a series of human immunodeficiency virus (HIV)-uninfected Chinese patients. In this case control study, a total of 251 patients with cryptococcosis and 464 healthy controls were included. One tag-single nucleotide polymorphism (SNP) (rs11045418) located at 5'-flanking region of Dectin-2 gene was selected and genotyped in this study. Among 251 patients, there were 108 (43%) meningitis patients including 73 (67.7%) healthy ones, 74 (29.5%) pulmonary infected patients including 49 (66.2%) healthy ones, and 69 (27.5%) patients with both neural and pulmonary infection including 38 (55.1%) immunocompetent ones. One hundred and forty-three (74 plus 69) patients with pulmonary cryptococcosis and 177 (108 plus 69) patients with cryptococcal meningitis were compared with controls, respectively. Three samples from 143 pulmonary infected patients failed in genotyping. There was a significant difference between 86 immunocompetent pulmonary infected patients and controls in the overdominant model (C/T vs. T/T + C/C; OR, 0.59; 95%CI, 0.37-0.94; P, .026). Similar but not significant difference was found between the overall pulmonary infected patients and the controls in the overdominant model (OR, 0.77; 95%CI, 0.52-1.12; P, .17). No such difference was found between controls and patients with cryptococcal meningitis. Our study firstly showed a genetic association between Dectin-2 and pulmonary cryptococcosis.

Evolution and transmission patterns of extensively drug-resistant tuberculosis in China.

The emergence and transmission of extensively drug-resistant tuberculosis (XDR-TB) pose an increasing threat to global TB control. This study aimed to identify the patterns of evolution and transmission dynamics of XDR-TB in populations in a region of China where TB is highly endemic. We analyzed a total of 95 XDR-TB isolates collected from 2003 to 2009 in Chongqing, China. Eight drug resistance genes covering 7 drugs that define XDR-TB were amplified by PCR followed by DNA sequencing. Variable-number tandem repeat 16-locus (VNTR-16) genotyping and genotypic drug resistance profiles were used to determine the evolution or transmission patterns of XDR-TB strains. Our results indicated that the Beijing genotype was predominant (85/95 [89.5%]) in XDR-TB strains, and as many as 40.0% (38/95) of the isolates were distributed into 6 clusters based on VNTR-16 genotyping and drug resistance mutation profiles. All isolates of each cluster harbored as many as six identical resistance mutations in the drug resistance genes rpoB, katG, inhA promoter, embB, rpsL, and gidB. Among the nine cases with continuous isolates from multidrug-resistant (MDR) to XDR-TB, 4 cases represented acquired drug resistance, 4 cases were caused by transmission, and 1 case was due to exogenous superinfection. The XDR-TB epidemic in China is mainly caused by a high degree of clonal transmission, but evolution from MDR to XDR and even superinfection with a new XDR strain can also occur.

Validation of thwaites' diagnostic scoring system for the differential diagnosis of tuberculous meningitis and bacterial meningitis.

To compare the clinical features of patients with tuberculous meningitis (TBM) and bacterial meningitis (BM) and to validate Thwaites' diagnostic scoring system for the differential diagnosis of TBM and BM, a retrospective review of 211 patients with TBM or BM who were admitted to Huashan Hospital, Fudan University, from 2007 to 2012 was conducted. The clinical characteristics and laboratory data were compared, and Thwaites' diagnostic scores were assessed at the time of admission for the differential diagnosis of TBM and BM. Significant differences were observed between the 2 groups in general information, clinical features, and cerebrospinal fluid characteristics. The sensitivity and specificity of Thwaites' diagnostic scoring system for the differential diagnosis of TBM and BM were found to be 98.2% and 43.6%, respectively, with positive and negative predictive values being 65.9% and 95.8%, respectively. The sensitivity and specificity for the differential diagnosis of TBM and initially treated BM were 98.2% and 82.9%, respectively, but were only 98.2% and 24.2% for that of TBM and partially treated BM, respectively. Thus, Thwaites' diagnostic scoring system was found to be highly effective for the differential diagnosis of TBM and initially treated BM but was found to be less effective for that of TBM and partially treated BM.

In vitro and in vivo evidence for amphotericin B as a P-glycoprotein substrate on the blood-brain barrier.

Amphotericin B (AMB) has been a mainstay therapy for fungal infections of the central nervous system, but its use has been limited by its poor penetration into the brain, the mechanism of which remains unclear. In this study, we aimed to investigate the role of P-glycoprotein (P-gp) in AMB crossing the blood-brain barrier (BBB). The uptake of AMB by primary brain capillary endothelial cells in vitro was significantly enhanced after inhibition of P-gp by verapamil. The impact of two model P-gp inhibitors, verapamil and itraconazole, on brain/plasma ratios of AMB was examined in both uninfected CD-1 mice and those intracerebrally infected with Cryptococcus neoformans. In uninfected mice, the brain/plasma ratios of AMB were increased 15 min (3.5 versus 2.0; P < 0.05) and 30 min (5.2 versus 2.8; P < 0.05) after administration of verapamil or 45 min (6.0 versus 3.9; P < 0.05) and 60 min (5.4 versus 3.8; P < 0.05) after itraconazole administration. The increases in brain/plasma ratios were also observed in infected mice treated with AMB and P-gp inhibitors. The brain tissue fungal CFU in infected mice were significantly lower in AMB-plus-itraconazole or verapamil groups than in the untreated group (P < 0.005), but none of the treatments protected the mice from succumbing to the infection. In conclusion, we demonstrated that P-gp inhibitors can enhance the uptake of AMB through the BBB, suggesting that AMB is a P-gp substrate.

Identification of clinically relevant fungi and prototheca species by rRNA gene sequencing and multilocus PCR coupled with electrospray ionization mass spectrometry.

BACKGROUND: Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse. METHODS: One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5' end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS. RESULTS: For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively. CONCLUSIONS: rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.

Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. ß-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.