A randomised Phase II trial to evaluate the toxicity of high-dose rifampicin to treat pulmonary tuberculosis.

SETTING: Randomised Phase IIB clinical trial. OBJECTIVES: To assess whether increasing the dose of rifampicin (RMP) from 10 mg/kg to 15 or 20 mg/kg results in an increase in grade 3 or 4 hepatic adverse events and/or serious adverse events (SAE). METHODS: Three hundred human immunodeficiency virus negative patients with newly diagnosed microscopy-positive pulmonary tuberculosis (TB) were randomly assigned to one of three regimens: 1) the control regimen (R10), comprising daily ethambutol (EMB), isoniazid (INH), RMP and pyrazinamide for 8 weeks, followed by INH and RMP daily for 18 weeks; 2) Study Regimen 1 (R15), as above, with the RMP dose increased to 15 mg/kg body weight daily for the first 16 weeks; and 3) Study Regimen 2 (R20), as above, with RMP increased to 20 mg/kg. Serum alanine transferase (ALT) levels were measured at regular intervals. RESULTS: There were seven grade 3 increases in ALT levels, 1/100 (1%) among R10 arm patients, 2/100 (2%) in the R15 arm and 4/100 (4%) in the R20 arm (trend test P = 0.15). One (R15) patient developed jaundice, requiring treatment modification. There were no grade 4 ALT increases. There was a non-significant increase in culture negativity at 8 weeks with increasing RMP dosage: 75% (69/92) in R10, 82.5% (66/80) in R15 and 83.1% (76/91) R20 patients (P = 0.16). CONCLUSIONS: No significant increase in adverse events occurred when the RMP dose was increased from 10 mg/kg to 15 mg/kg or 20 mg/kg.

Neurological, visual, and MRI brain scan findings in 87 South African patients with HIV-associated cryptococcal meningoencephalitis.

BACKGROUND: HIV-associated cryptococcal meningoencephalitis (CM) is a leading cause of adult meningitis in sub-Saharan Africa. Neuroradiological data is however limited to case reports and small case series from developed countries and/or immunocompetent patients. METHODS: Eighty seven patients aged ≥18 hospitalized with a first episode of CM had magnetic resonance (MRI) imaging during the first two weeks of admission. A subset of eleven patients had follow-up scans approximately one month from their initial MRI scan. All had prospectively-recorded detailed neurological and visual examinations. RESULTS: An abnormal finding on neurological examination was detected in 33 (39%) patients. 38 (48%) patients experienced some visual loss. Neuroradiological lesions presumed to be cryptococcosis-related, as defined by the presence of dilated Virchow Robin spaces, pseudocysts or cryptococcomas, enhancing nodules, hydrocephalus, meningitis, focal perilesional oedema and infarcts, were detected in 55 (63%) patients. MRI findings suggestive of a second diagnosis were found in 18 (21%) patients. Visual loss was associated with the presence of cryptococcal-related lesions (p = 0.02). Blindness was associated with raised intracranial pressure (ICP) (p = 0.02). Of eleven patients with paired scans, brain swelling was identified on the initial scan in only one patient. CONCLUSION: The majority of patients had MRI brain scan abnormalities presumed secondary to CM. Dilated Virchow Robin spaces were the commonest neuroradiological lesion. Visual loss was associated with the degree of cerebral involvement as reflected by the presence of MRI abnormalities. Blindness was associated with the presence of raised ICP. Initial generalised brain swelling does not appear to be common, but further studies with paired scans are needed.

Evaluation of a pro-active strategy for managing tuberculosis-HIV co-infection in a UK tertiary care setting.

Management of tuberculosis (TB)-HIV co-infection is complicated by interactions between the diseases and their therapies. We developed and evaluated a strategy to (i) treat co-infected patients in a single co-infection clinic, (ii) maximize use of first-line drugs, (iii) delay antiretroviral therapy (ART) until two months post-TB treatment except in severe immunosuppression, (iv) commence efavirenz at 600 mg daily with therapeutic drug monitoring (TDM) and (v) target treatment completion. We conducted a prospective cohort review over 5.5 years in a UK tertiary referral center where 56 HIV-positive patients treated for TB were followed-up for a median 30 months. Main outcome measures were treatment completion, adverse events, immune reconstitution inflammatory syndrome, immunological and virological parameters, and TDM for efavirenz. Treatment completion rates were 88% (49/56); four patients were lost to local follow-up and three (5.4%) died during treatment; no deaths were TB-related. Adverse events were common (55%), but caused no treatment interruptions. Standard doses (600 mg daily) of efavirenz with rifampicin achieved or exceeded therapeutic levels in 25/28 (89%). This study supports combined management for TB-HIV co-infected patients. Delaying ART to two months post-TB treatment did not seem to result in poor clinical outcomes in this well-resourced context. Although efavirenz 600 mg daily usually achieved satisfactory levels, TDM is recommended.

In vitro-clinical correlations for amphotericin B susceptibility in AIDS-associated cryptococcal meningitis.

Reliable measures of antifungal drug susceptibility are needed. We tested the susceptibility of Cryptococcus neoformans from patients treated with amphotericin B. In vitro susceptibility employed a modified broth macrodilution method. We demonstrate a strong correlation between the quantitative measures of in vitro amphotericin B susceptibility and the quantitative response observed in patients.

Determination of the pH of the Cryptococcus neoformans vacuole.

We have previously demonstrated the antifungal activity of the weak bases chloroquine and quinacrine against Cryptococcus neoformans. Quinacrine, being fluorescent, was seen to be concentrated within a complex vacuolar structure within the cryptococcal cell. Here we determined the pH of this compartment using the pH-sensitive fluorescent dye, 5-(and 6-) carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA). Carboxy-DCFDA was concentrated within the cryptococcal vacuole, giving a pattern of fluorescence similar to that previously observed with quinacrine. For each experiment, a standard curve of fluorescence ratio against pH was generated using buffers of defined pH containing a mixture of ionophores and inhibitors to equilibrate vacuolar pH with that of the medium. The pH of the cryptococcal vacuole of five strains was calculated to range from 5.3 to 5.9 with a mean of 5.6. This acidic pH is consistent with a model in which weak bases such as chloroquine and quinacrine are accumulated, by ion trapping within the fungal vacuole. Antifungal activity may result from the consequent disruption of pH-dependent processes as well as effects on other as yet undefined fungal targets.

Chloroquine and the fungal phagosome.

The antimalarial drug chloroquine accumulates inside the macrophage phagolysosome by ion trapping where it exerts potent antifungal activity against Histoplasma capsulatum and Cryptococcus neoformans by distinct mechanisms. Chloroquine inhibits growth of H. capsulatum by pH-dependent iron deprivation, whereas it is directly toxic to C. neoformans. Clearly, clinical studies are required to document the potential therapeutic efficacy of chloroquine or related congeners as adjuvant therapy in fungal disease. Moreover, the diversity of pathogenic microorganisms inhibited and/or killed by chloroquine makes this drug an attractive candidate for prophylactic therapy.

Conditional lethality of the diprotic weak bases chloroquine and quinacrine against Cryptococcus neoformans.

Chloroquine at 10 microM enhances the activity of macrophages against Cryptococcus neoformans but does not directly inhibit cryptococcal growth. The antifungal activity of higher chloroquine concentrations likely to be found within the acidic cryptococcal phagosome was tested. Concentrations of >/=30 microM inhibited cryptococcal growth, and there was fungal killing at concentrations of >/=100 microM. Activity was dependent on physiologic temperature and pH. Quinacrine was 50-fold more active than chloroquine, and concentrations as low as 100 nM enhanced macrophage anticryptococcal activity. Quinacrine was concentrated within a vacuolar system within the fungal cell and highly concentrated within intracellular C. neoformans. Ammonium chloride and bafilomycin A both inhibited cryptococcal growth, suggesting that the activity of chloroquine and quinacrine may in part be due to disruption of pH-dependent processes. These findings add to the known spectrum of activity of chloroquine and quinacrine. These, and related compounds, may have utility for the treatment of cryptococcosis.

Effect of interleukin (IL)-15 priming on IL-12 and interferon-gamma production by pathogen-stimulated peripheral blood mononuclear cells from human immunodeficiency virus-seropositive and -seronegative donors.

Hypoproduction of the cytokines interleukin (IL)-12 and interferon (IFN)-gamma is thought to contribute to the impaired immunity seen in human immunodeficiency virus (HIV)-infected persons. The effects of priming with IL-15 on the production of IL-12 and IFN-gamma by stimulated peripheral blood mononuclear cells (PBMC) from HIV-seronegative and -seropositive donors were studied. Stimuli included 3 pathogens that commonly infect HIV-positive persons-Cryptococcus neoformans, Candida albicans, and Mycobacterium tuberculosis-plus Staphylococcus aureus. Following IL-15 priming of HIV-negative PBMC, pathogen-stimulated IL-12 and IFN-gamma production increased 5-58-fold. However, for the HIV-positive PBMC, IL-15 priming did not lead to significant increases in pathogen-stimulated IL-12 production and caused only modest increases in IFN-gamma production. These data suggest that IL-15 alone may be insufficient to correct the defect in IL-12 and IFN-gamma production in HIV-positive persons.

Five scientists at Johns Hopkins in the modern evolution of neuroscience.

Neuroscience's evolution at Johns Hopkins, from neurophysiology to the new field of neurobiology, though unplanned, was rapid and important. Beginning in 1933 when Philip Bard became professor of physiology at Johns Hopkins, members of his department concentrated initially on neuroanatomical control of placing reactions and sexual activity. Vernon Mountcastle, extending available techniques, discovered vertical somato-sensory columns in the 1950's. Stephen Kuffler, who arrived at Hopkins in 1947, was a pioneer in single unit microelectrode recording techniques. He soon attracted scientists from all over the world to his laboratory. Among them, Torsten Wiesel and David Hubel discovered vertical neuronal columns in the visual cortex. During several decades at Johns Hopkins, these five scientists set extremely high scientific standards and established a climate of inquiry in which ideas were shared and young scientists encouraged. They contributed significantly to the emerging discipline of neuroscience.

Stool microflora in extremely low birthweight infants.

AIM: To serially characterise aerobic and anaerobic stool microflora in extremely low birthweight infants and to correlate colonisation patterns with clinical risk factors. METHODS: Stool specimens from 29 infants of birthweight <1000 g were collected on days 10, 20, and 30 after birth. Quantitative aerobic and anaerobic cultures were performed. RESULTS: By day 30, predominant species were Enterococcus faecalis, Escherichia coli, Staphylococcus epidermidis, Enterbacter cloacae, Klebsiella pneumoniae, and Staphylococcus haemolyticus. Lactobacillus and Bifidobacteria spp were identified in only one infant. In breast milk fed (but not in formula fed) infants, the total number of bacterial species/stool specimen increased significantly with time (2.50 (SE 0.34) on day 10; 3.13 (0.38) on day 20; 4.27 (0.45) on day 30) as did quantitative bacterial counts; Gram negative species accounted for most of the increase. On day 30, significant inverse correlations were found between days of previous antibiotic treatment and number of bacterial species (r=0.491) and total organisms/g of stool (r=0.482). Gestational age, birthweight, maternal antibiotic or steroid treatment, prolonged rupture of the membranes, and mode of delivery did not seem to affect colonisation patterns. CONCLUSIONS: The gut of extremely low birthweight infants is colonised by a paucity of bacterial species. Breast milking and reduction of antibiotic exposure are critical to increasing fecal microbial diversity.

Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages.

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2',7'-difluorofluorescein (Oregon Green). These probes have pKas of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 microM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.

Recent advances in cryptococcosis, candidiasis and coccidioidomycosis complicating HIV infection.

Concomitant with the decline in CD4+ T-cells seen as human immunodeficiency virus (HIV) infection progresses, the prevalence of opportunistic mycoses increases dramatically. This article reviews selected recent advances in our understanding of the immunology, molecular epidemiology and treatment of fungal infections in patients infected with HIV. For cryptococcosis, studies are reported on how HIV infection affects the immune response to Cryptococcus neoformans and, conversely, how stimulation with C. neoformans induces HIV production from latently HIV-infected cells. In addition, studies are presented examining the efficacy of triple combination antimycotic chemotherapy in cryptococcosis. For candidosis, investigations into genetic profiles of Candida albicans isolates obtained from patients, with resistance to antifungal agents, are demonstrated. Finally, for coccidioidomycosis, prospective studies are presented examining the clinical, epidemiological and immunological characteristics of a cohort of HIV-infected subjects residing in an endemic area.

Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation.

Infections due to Cryptococcus neoformans are common in AIDS patients. We investigated the effect of chloroquine, which raises the pH of phagolysosomes, on the anticryptococcal activity of mononuclear phagocytes. C. neoformans multiplied within monocyte-derived macrophages (MDM) in the absence of chloroquine but were killed with the addition of chloroquine. Ammonium chloride was also beneficial, suggesting that effects were mediated by alkalinizing the phagolysosome. Chloroquine inhibits growth of other intracellular pathogens by limiting iron availability. However, chloroquine-induced augmentation of MDM anticryptococcal activity was unaffected by iron nitriloacetate, demonstrating that chloroquine worked by a mechanism independent of iron deprivation. There was an inverse correlation between growth of C. neoformans in cell-free media and pH, suggesting that some of the effect of chloroquine on the anticryptococcal activity of MDM could be explained by relatively poor growth at higher pH. Chloroquine enhanced MDM anticryptococcal activity against all tested cryptococcal strains except for one large-capsule strain which was not phagocytosed. Positive effects of chloroquine were also seen in monocytes from both HIV-infected and -uninfected donors. Finally, chloroquine was therapeutic in experimental cryptococcosis in outbred and severe combined immunodeficient mice. Thus, chloroquine enhances the activity of mononuclear phagocytes against C. neoformans by iron-independent, pH-dependent mechanisms and is therapeutic in murine models of cryptococcosis. Chloroquine might have clinical utility for the prophylaxis and treatment of human cryptococcosis.

Induction of human immunodeficiency virus type 1 expression in monocytic cells by Cryptococcus neoformans and Candida albicans.

Because candidiasis and cryptococcosis are common in human immunodeficiency virus (HIV)-infected persons, the effect of Cryptococcus neoformans and Candida albicans on HIV expression in monocytic cells was examined. Stimulation of the latently HIV-infected myelomonocytic cell line OM-10.1 with C. neoformans and C. albicans in the presence of pooled human serum caused a ratio-dependent increase in HIV production. Induction of HIV by C. neoformans was enhanced by anti-capsular antibody, while induction by both organisms was inhibited by anti-TNF-alpha antibody. In THP-1 cells transfected with HIV plasmid constructs, both organisms induced transcription from the HIV long terminal repeat that was dependent on intact NF-kappaB binding sequences. Thus, C. neoformans and C. albicans enhance HIV expression in monocytic cells through a TNF-alpha- and NF-kappaB-dependent mechanism. In HIV-infected patients, such enhancement may further impair host immunity and could accelerate the course of HIV disease.

Mechanisms of impaired anticryptococcal activity of monocytes from donors infected with human immunodeficiency virus.

The mechanisms by which monocytes from patients infected with human immunodeficiency virus (HIV) have reduced growth inhibitory activity against Cryptococcus neoformans was examined. Monocyte-enriched peripheral blood mononuclear cells from 12 HIV-seropositive donors with CD4 cell counts of 10-210 cells/mm3 (median, 85) and HIV-seronegative donors were compared in assays to determine the binding and phagocytosis of C. neoformans and the respiratory burst and degranulation in response to C. neoformans and zymosan. Monocytes from HIV-infected and uninfected persons bound and ingested C. neoformans equally well; however, generation of hydrogen peroxide and specific release of beta-glucuronidase in response to C. neoformans was significantly reduced in monocyte-enriched cells from the HIV-infected donors. The impaired anticryptococcal activity of monocytes from persons with HIV may be related to defects in both oxidative and nonoxidative effector pathways that occur after the binding and internalization of the organism.

Variables affecting production of monocyte chemotactic factor 1 from human leukocytes stimulated with Cryptococcus neoformans.

The chemokine monocyte chemoattractant protein 1 (MCP-1) is produced predominantly by mononuclear phagocytes and stimulates recruitment into infected tissues of blood monocytes and T cells. These cell types are thought to be critical to host defenses against infections due to Cryptococcus neoformans, a major cause of disease in persons with AIDS and other disorders of cell-mediated immunity. Accordingly, in the present study, we examined the conditions under which human monocytes and bronchoalveolar macrophages (BAM) are stimulated by C. neoformans to produce MCP-1. C. neoformans was a potent inducer of MCP-1 release from monocytes, with levels of chemokine secreted similar to that seen following stimulation with lipopolysaccharide (LPS). BAM, in contrast, were stimulated by LPS, but not by C. neoformans, to secrete MCP-1. A peak in MCP-1 mRNA was seen 8 h following cryptococcal stimulation of monocytes. Nine strains of C. neoformans stimulated monocytes to release MCP-1, and there was only modest variation between strains. However, when an individual strain was used, the capacity of C. neoformans to stimulate monocyte MCP-1 release did vary, depending upon the conditions used to grow the fungal stimuli. Finally, C. neoformans stimulated comparable quantities of MCP-1 release in monocytes from donors with and without human immunodeficiency virus infection. These data establish C. neoformans as a potent stimulator of MCP-1 in monocytes, but not in BAM. The failure of C. neoformans to stimulate MCP-1 in BAM, if occurring in vivo, could result in a diminished cell-mediated inflammatory response following inhalation of airborne fungi.

Priming with IFN-gamma restores deficient IL-12 production by peripheral blood mononuclear cells from HIV-seropositive donors.

Production of IL-12 is deficient in PBMC from HIV-infected individuals. Because of recent studies demonstrating that IFN-gamma priming increases the production of IL-12 in normal PBMC, we examined the role of IFN-gamma in the production of IL-12 in PBMC from HIV-seropositive donors. In response to Staphylococcus aureus, production of IFN-gamma and IL-12 was reduced in PBMC from HIV-seropositive compared with that from HIV-seronegative donors. Priming with IFN-gamma, through increases in both IL-12 p40 and p35 mRNA levels, caused a significant increase in IL-12 release by PBMC from both HIV-seropositive and HIV-seronegative donors. However, the increase was greater for PBMC from HIV-seropositive donors, largely restoring the deficit in IL-12 production seen in unprimed cells. In response to Cryptococcus neoformans, Candida albicans, and Mycobacterium tuberculosis, three pathogens that frequently cause opportunistic infections in persons with AIDS, IFN-gamma production was also reduced in PBMC from HIV-seropositive compared with seronegative donors. When primed with IFN-gamma, PBMC from both HIV-seropositive and seronegative donors released substantial and similar quantities of IL-12 in response to these organisms. Taken together, these results demonstrate that IFN-gamma can restore the deficit in IL-12 production seen in HIV infection.