Epidemiology of meningitis with a negative CSF Gram stain: under-utilization of available diagnostic tests.

Meningitis with a negative cerebrospinal fluid Gram stain (CSF-GS) poses a diagnostic challenge as more than 50% of patients remain without an aetiology. The introduction of polymerase chain reaction (PCR) and arboviral serologies have increased diagnostic capabilities, yet large scale epidemiological studies evaluating their use in clinical practice are lacking. We conducted a prospective observational study in New Orleans between November 1999 and September 2008 (early era) when PCR was not widely available, and in Houston between November 2008 and June 2013 (modern era), when PCR was commonly used. Patients presenting with meningitis and negative CSF-GS were followed for 4 weeks. All investigations, PCR used, and results were recorded as they became available. In 323 patients enrolled, PCR provided the highest diagnostic yield (24·2%) but was ordered for 128 (39·6%) patients; followed by serology for arboviruses (15%) that was ordered for 100 (31%) of all patients. The yield of blood cultures was (10·3%) and that of CSF cultures was 4%; the yield for all other tests was <10%. Overall, 65% of the patients remained without a diagnosis at 4 weeks: 72·1% in early era vs. 53·4% (P < 0·01) in modern era; this change was attributed to diagnosing more viral pathogens, 8·3% and 26·3% (P < 0·01), respectively. The introduction of PCR and arboviral serologies has improved the yield of diagnosing patients with meningitis and a negative CSF-GS, but both tests are being under-utilized.

Evaluation of antifungal therapy in patients with candidaemia based on susceptibility testing results: implications for antimicrobial stewardship programmes.

BACKGROUND: Definitive antifungal therapy is typically based on Candida species and clinical status, rather than susceptibility reports. Antifungal susceptibility testing is available, but the impact on treatment decisions is unknown. The purpose of this study was to assess antifungal therapy in hospitalized patients with candidaemia during the time period between the start of empirical therapy and after antifungal susceptibility testing reports are available. METHODS: A retrospective study of 161 hospitalized patients with candidaemia was conducted. Patients who received fluconazole or an echinocandin were evaluated for changes in empirical antifungal therapy prior to and after susceptibility reporting. RESULTS: One hundred and sixty-one patients aged 59 ±â€Š16 years (male, 54%; Caucasian, 52%; APACHE II score ≥ 15, 48%; and intensive care unit, 50%) were identified, of whom 130 (81%) had fluconazole-susceptible candidaemia. Fifty-eight patients (36%) were initiated on fluconazole and 103 (64%) on an echinocandin. The mean time from culture to the susceptibility report was 5 ±â€Š2 days. Prior to availability of the susceptibility report, 20 fluconazole-initiated patients (34%) were switched to an echinocandin, while 14 echinocandin-initiated patients (14%) were switched to fluconazole. Once a susceptibility report was available, 35 of 89 (39%) patients with fluconazole-susceptible candidaemia on an echinocandin were de-escalated to fluconazole. Eleven patients on fluconazole just prior to a susceptibility report were identified with a fluconazole-resistant Candida species. CONCLUSIONS: Using antifungal susceptibility testing, patients given fluconazole with fluconazole-resistant Candida species were identified. Less than 40% of echinocandin-treated patients with fluconazole-susceptible organisms were de-escalated to fluconazole. Antifungal susceptibility testing may help to identify patients in need of clinical intervention.

Vaccine-induced cytokine responses in a guinea pig model of pulmonary tuberculosis.

Guinea pigs exposed to very small numbers of virulent tubercle bacilli by the respiratory route develop a disease which mimics many of the important features of the pathogenesis of human tuberculosis (TB), including the expression of strong protective immunity following vaccination with BCG. In order to elucidate the precise immunological mechanisms of vaccine-induced resistance in this model, both mRNA and protein assays for several guinea pig cytokines and chemokines have been developed. The coordinated expression of cytokine and chemokine mRNA and protein was examined in various leukocyte populations and in inflammatory cells and fluid collected following the induction of tuberculous pleurisy in BCG-vaccinated guinea pigs. Real-time RT-PCR assays revealed that the mRNA levels for IFNgamma, TNFalpha, and IL-8 rose over the first few days of TB pleuritis and then declined over the 9 days of the study. Injection of anti-TGFbeta on day 8 following pleurisy induction resulted in significant changes in cytokine mRNA levels and PPD-induced proliferation in pleural effusion lymphocytes taken 24h later. BCG vaccination induced significantly higher levels of bioactive TNFalpha protein in the supernatants of alveolar, peritoneal and splenic cells from BCG-vaccinated guinea pigs cultured in the presence of attenuated or virulent mycobacteria. In sharp contrast, following virulent challenge, all three cell types from BCG-vaccinated guinea pigs produced significantly less TNFalpha. Thus, BCG vaccination appears to modulate the potentially harmful effects of TNFalpha in this model of pulmonary TB. Levels of mRNA for IL-12p40 were upregulated by exposure of infected and uninfected macrophages to recombinant guinea pig (rgp)TNFalpha. The intracellular survival of mycobacteria was enhanced when endogeous TNFalpha activity was neutralized with anti-rgpTNFalpha antiserum. rgp RANTES (CCL5) upregulated mRNA levels for TNFalpha, IL-1beta, MCP-1 (CCL2), and IL-8 (CXCL8) in alveolar and peritoneal macrophages. These results illustrate the profound effects of prior vaccination with BCG on the cytokine and chemokine responses of distinct cell populations in the guinea pig following exposure to attenuated and virulent strains of M. tuberculosis.

Production and characterization of guinea pig recombinant gamma interferon and its effect on macrophage activation.

Gamma interferon (IFN-gamma) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-gamma (gpIFN-gamma) and reported that BCG vaccination induced a significant increase in the IFN-gamma mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain of Mycobacterium tuberculosis stimulated less IFN-gamma mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-gamma with a histidine tag at the N terminus (His-tagged rgpIFN-gamma) in Escherichia coli. rgpIFN-gamma was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-gamma. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-gamma was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-gamma did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-gamma induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-gamma has been successfully produced and characterized in our laboratory. The study of rgpIFN-gamma will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.

IroN, a novel outer membrane siderophore receptor characteristic of Salmonella enterica.

Speciation in enterobacteria involved horizontal gene transfer. Therefore, analysis of genes acquired by horizontal transfer that are present in one species but not its close relatives is expected to give insights into how new bacterial species were formed. In this study we characterize iroN, a gene located downstream of the iroBC operon in the iroA locus of Salmonella enterica serotype Typhi. Like iroBC, the iroN gene is present in all phylogenetic lineages of S. enterica but is absent from closely related species such as Salmonella bongori or Escherichia coli. Comparison of the deduced amino acid sequence of iroN with other proteins suggested that this gene encodes an outer membrane siderophore receptor protein. Mutational analysis in S. enterica and expression in E. coli identified a 78-kDa outer membrane protein as the iroN gene product. When introduced into an E. coli fepA cir fiu aroB mutant on a cosmid, iroN mediated utilization of structurally related catecholate siderophores, including N-(2,3-dihydroxybenzoyl)-L-serine, myxochelin A, benzaldehyde-2,3-dihydroxybenzhydrazone, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-L-lysine, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-L-lysine amide, and enterochelin. These results suggest that the iroA locus functions in iron acquisition in S. enterica.