Effect of increasing inoculum sizes of Aspergillus hyphae on MICs and MFCs of antifungal agents by broth microdilution method.

In order to investigate the influence of different hyphal inoculum sizes on minimal inhibition concentrations (MICs) and minimum fungicidal concentrations (MFCs) of amphotericin B (AMB), voriconazole and itraconazole, five isolates each of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus were studied using a broth microdilution method. Three inoculum sizes were used: 1 x 10(3)-5 x 10(3), 1 x 10(4)-5 x 10(4) and 1 x 10(5)-5 x 10(5) cfu/ml. MICs and MFCs were read at 24 and 48 h at 35 degrees C. For all species tested, AMB MICs and MFCs were minimally affected by inoculum size on. However inoculum size significantly affected MICs and MFCs of voriconazole and itraconazole; there was an increase of up to 6-fold in MICs and MFCs for the various aspergilli when the inoculum increased from 10(3) to 10(5) cfu/ml (P<0.05). Thus azoles showed significant inoculum effects, while AMB showed comparatively minimum inoculum effects against Aspergillus spp.

Visceral leishmaniasis in an AIDS patient on successful antiretroviral therapy: failure of parasite eradication despite increase in CD4+ T-cell count but low CD8+ T-cell count.

An unusual cutaneous relapse of visceral leishmaniasis (initially mistaken for eruptive histiocytomas) was seen in an AIDS patient despite good virological and CD4+ T-cell responses to highly active antiretroviral therapy. Splenectomy and the patient's low CD8+ T-cell count are discussed as possible causes of failed disease control.

Complications after laryngeal surgery: videofluoroscopic evaluation of 120 patients.

AIM: Videofluoroscopic assessment of the spectrum and incidence of swallowing complications after state-of-the-art laryngeal cancer surgery. MATERIALS AND METHODS: We retrospectively studied videofluoroscopic examinations of 120 patients (94 men, 26 women; mean age, 58 years) with suspected complications after laryngeal resection (partial laryngectomy, 65; total laryngectomy, 55). Swallowing function (i.e., oral bolus control, laryngeal elevation and closure, presence of pharyngeal residue, aspiration) and structural abnormalities such as strictures, fistulas and tumour recurrence were assessed by videofluoroscopy. RESULTS: Abnormalities were found in 110 patients, including strictures in nine, fistulas in six and mass lesions in 13 patients. Aspiration was found in 63 patients overall (partial laryngectomy, 61/65; total laryngectomy, 2/55), occurring before swallowing in five, during swallowing in 34, after swallowing in nine and at more than one phase in 15 patients. Pharyngeal paresis was detected in three and pharyngeal weakness in 19 patients. Pharyngo-oesophageal sphincter dysfunction was observed in 10 cases. CONCLUSION: Aspiration is a very common complication after partial laryngeal resection. It is mainly caused by incomplete laryngeal closure, sphincter dysfunction or pharyngeal pooling. Videofluoroscopy is the only radiological technique able to identify both disordered swallowing function and structural changes after laryngeal resection. Detection of these complications is crucial for appropriate further therapy.Kreuzer, S. H. (2000). Clinical Radiology55, 775-781.

Aspergillus terreus infections in haematological malignancies: molecular epidemiology suggests association with in-hospital plants.

During a three-year period nine patients with haematological diseases after myeloablative chemotherapy died from invasive fungal infections caused by Aspergillus terreus. The hospital inanimate environment was monitored and A. terreus was cultured from potted plants in the vicinity of the patients. The patients (N = 14) and the environmental isolates (N = 2) were fingerprinted by RAPD-PCR with four different primers. Based on RAPD patterns the patients' isolates were differentiated into five different types; the environmental isolates represented two types. The isolates of four patients were identical to those found in the environment. Five additional patients were infected by RAPD types not found in the environment. One patient was infected with two different types. The data indicate a hospital-acquired infection in many of the patients and underline the need for careful environmental monitoring of units in which high-risk patients are housed.

Dissimilar attenuation of Candida albicans virulence properties by human immunodeficiency virus type 1 protease inhibitors.

The secreted aspartyl proteinase (Sap) of Candida albicans, which is believed to represent an important virulence factor of this opportunistic yeast, and the human immunodeficiency virus type 1 (HIV-1) protease, which is obligatory for the production of infectious virions, both belong to the same family of aspartyl proteinases. We have previously shown that the HIV-1 protease inhibitor Indinavir directly inhibits secretion and proteinase activity of Sap in a dose-dependent manner. Furthermore, at very high concentrations, viability of C. albicans is markedly reduced by Indinavir, indicating that HIV-1 protease inhibitors may possess antifungal activity. We thus proposed that these drugs may add to the resolution of mucosal candidiasis in HIV-1 infected subjects. We have now compared three different HIV-1 protease inhibitors. The rank order of Sap inhibition, already significant at 0.1 mg/ml for all protease inhibitors, was Ritonavir > Indinavir > Saquinavir. However, the cross-reactivity of Ritonavir to pepsin was also more pronounced compared with the other two. Indinavir did not affect Candida viability at concentrations up to 1 mg/ml, in line with our previous study. In contrast, at this concentration Saquinavir was even fungicidal as assessed by three different viability assays (colony formation assay, MTT assay, propidium iodide staining) whereas Ritonavir significantly affected the mitochondrial activity only (MTT assay). No influence on Candida viability was observed for any of the three at concentrations of 0.1 mg/ml or lower. It remains to be examined whether HIV-1 protease inhibitors or derivatives thereof may be suitable for in vivo therapy of subjects suffering from mucosal candidiasis resistant to current antimycotics.

In-vitro testing of susceptibility to amphotericin B is a reliable predictor of clinical outcome in invasive aspergillosis.

Invasive aspergillosis is a life-threatening fungal infection which, in neutropenic patients, is associated with an extremely high mortality rate despite optimal treatment. In order to investigate microbiological risk factors for treatment failures in more detail, Aspergillus spp. obtained from 29 patients with haematological diseases after myelo-ablative chemotherapy and bone marrow transplantation were analysed for their susceptibility to amphotericin B in vitro and this was compared with clinical outcome to see if there was a correlation. Aspergillus flavus was present in 12 (41 %) of the 29 patients, Aspergillus terreus in nine (31%) and Aspergillus fumigatus in eight (28%). The susceptibility of these isolates to amphotericin B varied between and within the three species. A. terreus was the only organism against which the MIC was consistently high, A. fumigatus and A. flavus showing variation between isolates in the degree of resistance to amphotericin B. The degree of in-vitro resistance was the only parameter correlating with clinical outcome in a univariate analysis and the only prognostic value in a multivariate analysis considering known risk factors. Irrespective of the species, all six patients with isolates against which the MIC was <2 mg/L survived, whereas most (22/23) of those with isolates resistant to > or = 2 mg/L died. Infections among the six survivors were caused by amphotericin B-susceptible A. fumigatus and A. flavus, but not A. terreus. We conclude that the outcome of aspergillus infection depends on the in-vitro susceptibility of the isolates to amphotericin B. Survival was poor in patients with isolates resistant to amphotericin B and good in those with amphotericin B-susceptible specimens. A. terreus was always associated with high resistance to amphotericin B and with poor survival.

Human B-cell TNF-beta microheterogeneity.

The production of TNF-alpha and TNF-beta by human B-cell lines was studied at both the molecular and biological levels. The 24 B-cell lines studied included EBV+ cell lines (n = 13), EBV- cell lines (n = 8), and AIDS-associated B-cell lines (AABCL) (n = 3) which are EBV+/HIV-. Whereas radioimmunoprecipitation using TNF-alpha antisera detected 17-kDa TNF-alpha as expected, similar studies with anti-TNF-beta antisera revealed TNF-beta microheterogeneity. In the AABCL three bands with approximate MW of 26, 24, and 22 kDa were detected under reducing conditions, and in the non-AABCL, two bands only with 26 and 22 kDa were observed. To determine whether the size heterogeneity of TNF-beta is due to glycosylation, TNF-beta deglycosylation studies were done in two AABCL (PA682BM-2, PA682PE-1) and one non-AABCL (IM-1178). As control, the normal lymphoblastoid B-cell line RPMI-1788, which is known to secrete TNF-beta with MW 25 and 20 kDa, has been used. Deglycosylation studies using N-glycanase + neuraminidase + O-glycanase reduced the various bands in all cell lines to one band with 18.6 kDa, which is compatible with the TNF-beta backbone. In attempt to determine whether the differential glycosylation of TNF has any functional significance, all 24 cell lines were studied for TNF secretion and for TNF neutralization by monoclonal antibodies and polyclonal antibodies to TNF-alpha and TNF-beta. Constitutive secretion of TNF-alpha and TNF-beta has been detected only in the three AABCL. Following activation with the tumor promoter teleocidin, the secretion of both TNFs has been triggered in 2/8 EBV- cell lines and in 8/13 EBV+ non-AABCL. Using rabbit polyclonal antibodies to human TNF-alpha and to human TNF-beta, only little if any neutralization of these TNFs has been shown. Our data suggest that the differences in glycosylation of B-cell-derived TNFs may account for the incomplete neutralization, and may influence the cytotoxic biological activity of this lymphokine.

Size heterogeneity of human lymphotoxin is due to O-linked glycosylation.

Lymphotoxin (LT), a cytokine with antiviral and antitumor activities, is produced by activated T- and B-lymphocytes. The molecular weight (MW) of this glycoprotein has been reported to be 24 and 25 kDa for the mature molecule and 18.6 kDa for the recombinant form. Here we report that various human T- and B-cell lines as well as freshly stimulated T and B cells release LT molecules of different sizes, ranging from 23 to 27 kDa by SDS polyacrylamide gele electrophoresis (PAGE) analysis. Although individual cell lines produce LT of characteristic size, no firm association between the different MW forms with either the B or the T cell lineage could be established. The size heterogeneity of LT is due to O-linked glycosylation since only the removal of both N- and O-linked sugar residues but not removal of N-linked sugar residues alone, reduces the size of all forms to around 18.6 kDa, which corresponds to the calculated MW of the recombinant form of human LT.