In vitro susceptibility testing of Candida and Aspergillus spp. to voriconazole and other antifungal agents using Etest: results of a French multicentre study.

Minimum inhibitory concentrations (MICs) of the antifungal agent voriconazole were determined using the Etest and compared with those of amphotericin B, itraconazole and fluconazole using 1986 clinical isolates of Candida spp. Voriconazole MICs were also compared with those of amphotericin B and itraconazole using 391 clinical isolates of Aspergillus spp. Voriconazole was found to have more potent activity and lower MIC values than amphotericin B, itraconazole and fluconazole against C. albicans, C. tropicalis, C. parapsilosis and C. kefyr. Against C. glabrata and C. krusei, voriconazole was more active than either of the other two azole antifungals but had similar activity to amphotericin B. For species of Aspergillus, MIC values of voriconazole were lower than those of amphotericin B and itraconazole against A. fumigatus and A. flavus, and were similar to those of amphotericin B against A. niger. Against A. terreus, MIC values for voriconazole and itraconazole were similar. A. terreus is known to be resistant to amphotericin B, and this was reflected in higher MIC values compared with those of voriconazole and itraconazole. Voriconazole therefore compares very favourably with other antifungal agents against a large number of clinical isolates of Candida and Aspergillus spp.

A novel antibody overlay technique for two-dimensional immunoelectrophoresis.

A new antibody overlay technique for 2-dimensional immunoelectrophoresis (2-D-IEP) is described. After the first dimension electrophoresis of the antigen, the desired amount of antiserum is applied to the initial agarose layer and then evenly distributed over a defined surface of the gel with a PVC film. This modification of the conventional 2-D-IEP procedure makes it possible to perform tandem 2-D-IEPs comparing 2 antisera on the same gel plate, rocket IEPs where several antisera are compared by electrophoresis on the same gel plate, and 2-D-IEPs with an intermediate antiserum, avoiding the need to pour an intermediate gel. With this technique, 77 antigens have been demonstrated in a Candida albicans serotype A somatic antigen preparation. This is the first description of such a large number of immunoprecipitates on the same immunoplate.

In vitro antimalarial activity of vegetal extracts used in West African traditional medicine.

Among strategies for the development of new antimalarials, a study of plants traditionally used in Africa against malaria has been pursued. Extracts obtained from the plants Azadirachta indica, Cinnamonum camphora, Lippia multiflora, Vernonia colorata, Guiera senegalensis, Combretum micranthum, and Ximenia americana, commonly used in Cote d'Ivoire by native healers for the treatment of malaria, were tested on two strains of Plasmodium falciparum: FcB1-Colombia (chloroquine-resistant) and F32-Tanzania (chloroquine-sensitive). Extracts were obtained after infusion and decoction, both techniques being used by most native healers. The antimalarial activities of the extracts were tested first by parasite 3H-hypoxanthine incorporation and second by visual evaluation of the activities of plant extracts on thin blood smears, which also permitted the determination of parasitic stages and parasite alteration. Among the seven plants tested, some had an apparent inhibitory effect on P. falciparum growth in vitro, while other seemed to be less efficient.

Clonality structure in Candida dubliniensis.

Multilocus enzyme electrophoresis was performed on 76 European strains of Candida dubliniensis. Ten of the 20 enzyme-encoding loci were polymorphic, giving rise to 10 electrophoretic types within the sample studied. Investigation of the population genetics of a subset of 36 strains from HIV-infected patients in London showed the existence of strong heterozygote deficits and excesses associated with significant linkage disequilibria between pairs of loci. These findings, together with the predominance of multilocus genotypes, strongly suggest that C. dubliniensis is mainly (if not totally) clonal. Analysis of genotypes of a larger number of strains should confirm this conclusion and improve our understanding of the epidemiology of this pathogen.

Simultaneous carriage of Candida albicans strains from HIV-infected patients with oral candidiasis: multilocus enzyme electrophoresis analysis.

Genetic diversity of 160 Candida albicans isolates from the oral cavity of 16 HIV-infected adults prior to antifungal treatment was assessed using multilocus enzyme electrophoresis (10 C. albicans colonies were randomly chosen from each specimen culture). 20 electrophoretic types were distinguished from the analysis of 21 enzyme loci (10 were polymorphic). Five patients (31%) were found to be colonized by 2 or 3 genetically distinct strains. Nevertheless, in these five cases, one strain predominated (from 7 to 9 of the 10 colonies). Some HIV + patients with oral candidiasis appear to be simultaneously infected with several genetically different C. albicans strains before antifungal treatment.

Multilocus enzyme electrophoresis analysis of Aspergillus fumigatus strains isolated from the first clinical sample from patients with invasive aspergillosis. EBGA Network. European Research Group on Biotype and Genotype of Aspergillus.

The genotypes of 50 isolates of Aspergillus fumigatus from 11 patients with invasive aspergillosis, obtained from three hospitals in different geographical areas, were determined by multilocus enzyme electrophoresis (MLEE). The study analysed the genetic polymorphism of multiple isolates from the first sample. Seven of the 14 enzymic loci studied were polymorphic, giving rise to eight different electrophoretic types. For nine of 11 patients studied, no polymorphism was observed in isolates within the first clinical sample. Analysis of genetic distance between electrophoretic types demonstrated a genetic heterogeneity within each geographical site. Moreover, some genotypes were preferentially found in a given area and this revealed a population structure within these geographical sites. Therefore, the epidemiology of A. fumigatus should be considered separately for each of these areas. The multiple discriminatory markers of MLEE seem to provide a powerful tool for increasing the understanding of the biology of this fungus.

Combination of three typing methods for the molecular epidemiology of Aspergillus fumigatus infections. European Research Group on Biotype and Genotype of Aspergillus.

This study investigated the source of infection and strain relatedness of Aspergillus fumigatus isolates from bronchial colonisation and invasive aspergillosis (IA) in four transplant patients. Environmental isolates from the patient's home and from the hospital and infecting isolates were obtained for patient A who developed IA. Clinic environmental and colonising isolates were obtained for patient B. Sequential isolates were obtained from various organs from patient C who developed IA and also from patient D who had a bronchitic aspergillosis that developed into IA. Ninety-one A. fumigatus isolates were analysed by three typing methods: multi-locus enzyme electrophoresis (MLEE), random amplified polymorphic DNA (RAPD) and sequence-specific DNA primers (SSDP). The three combined typing methods demonstrated a greater differentiation of isolates than the typing methods used separately or in pairs. This demonstrated the genotypic variability of A. fumigatus and facilitated better epidemiological analysis. Large polymorphisms were demonstrated for each patient isolate between and colonies within various samples. The relatedness of the isolates suggested nosocomially acquired aspergillosis for patient B, but the source of infection for patient A remained unclear. The results suggested at least three multiple infections among the four patients. This study enabled the identification of the source of infection and strain relatedness, which in turn facilitates the development of preventive measures for patient management in the future.

Antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I.

An antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I (VHSV I) was developed. The assay employs two monoclonal antibodies (mAb) directed against distinct epitopes of the viral envelope glycoprotein (Gp). The antigen bound by the capture mAb (A17) was detected by addition of a second mAb (L7) conjugated to horseradish peroxidase, followed by addition of the enzyme substrate. The technique is highly sensitive, enabling detection of the virus at a protein concentration as low as 1 ng/ml of total proteins (1.5 fmol of envelope Gp per ml) in purified preparations. VHSV I was also detected in culture supernatants (5 x 10(5) PFU/ml) and in extracts of kidney and spleen of rainbow trout infected experimentally (5 x 10(5) PFU/ml). The assay was highly specific: infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus, spring viraemia of carp virus, pike fry rhabdovirus, eel rhabdovirus and perch rhabdovirus could not be detected by the antigen-capture ELISA for VHSV I.

Molecular typing of Aspergillus fumigatus strains by sequence-specific DNA primer (SSDP) analysis.

A PCR typing method has been developed and tested to investigate the polymorphism of clinical strains of Aspergillus fumigatus. Firstly, the DNA fragments from random amplified polymorphic DNA (RAPD) patterns of nine epidemiologically and geographically non-related monosporal strains of A. fumigatus were cloned and sequenced. The pairs of five sequence-specific DNA primers (SSDP), characteristic of the 5' and 3' extremities of the RAPD products, were then used in high stringency PCR to type 43 clinical strains of A. fumigatus from 13 patients, according to the presence or absence of a single amplified band. This original approach, which uses the advantages of PCR, has made it possible to overcome the difficulties resulting from the low stringency amplification. The SSDP analysis of 51 A. fumigatus strains (9 unrelated monosporal strains and 43 clinical strains from 13 patients) can be classed into 22 different types with a high reproducibility and a high level of discrimination (D = 0.96). The results suggest that seven lung transplant patients with necrotizing aspergillosis, bronchitis aspergillosis and bronchial colonization were infected by multiple strain genotypes, whereas three patients with invasive aspergillosis seem to have been infected by a single strain.

Composition and antimalarial activity in vitro of volatile components of Lippia multiflora.

The essential oil of Lippia multiflora was prepared by hydrodistillation of leaves and stalks and characterized by GC and mass spectroscopy. The oil was tested for antimalarial activity on in vitro cultures of Plasmodium falciparum (FcB1-Columbia chloroquine-resistant strain and F32-Tanzania chloroquine-sensitive strain). The dilutions inhibiting the in vitro growth of the parasite by 50% 24 and 72 hr after administration of the essential oil to the parasite culture were 1/12,000 and 1/21,000, respectively. When tested on a highly synchronized culture, the essential oil inhibited growth mostly at the trophozoite-schizont step, indicating a potential effect on the first nuclear division of the parasite.

Antimalarial activity in vitro of Cochlospermum tinctorium tubercle extracts.

Resistance of Plasmodium falciparum to current antimalarial compounds has drastically increased during the last few years and is now a major public health problem. We have studied plants traditionally used in Africa against malaria. Extracts of the tubercles of Cochlospermum tinctorium A. Rich, commonly used in Burkina Faso, were tested in vitro on 2 strains of P. falciparum, one (FcB1-Colombia) chloroquine resistant and the other (F32-Tanzania) chloroquine sensitive. Extracts were obtained by infusion and decoction. The 50% inhibitory concentrations (IC50) were determined by measuring [3H]hypoxanthine incorporation and also by microscopical examination which permitted the determination of parasite stages. We obtained similar results with fresh extracts, frozen extracts, and lyophilized extracts of C. tinctorum. IC50 values were of the order of 1-2 micrograms/mL, about one-tenth of those reported for extracts of neem leaves (Azadirachta indica) and about half the values reported for Artemisia annua extracts.

Case report of three Candida albicans infections detected at delivery.

We report three similar cases of Candida albicans infections in neonates, at delivery. A retrospective study of the isolates was conducted to define the diversity of infective strains and their susceptibility to amphotericin B and fluconazole. Three neonates with fever, 'not doing well' at delivery had positive cultures for C. albicans. Samples were then taken from the mothers who did not exhibit any clinical symptoms of infection. Candida albicans strains isolated from both neonates and mothers were cultured, six colonies of each were typed by multilocus enzyme electrophoresis. The E-test method was used to determine the susceptibility of each colony to the two antifungals commonly used in this unit: amphotericin B and fluconazole. The initial isolates were composed of different types of strains. In the three cases, one of the mother types was found in the neonate isolates, leading us to suggest a vertical transmission of strains. All of the other types were distinct. All of the types were susceptible to amphotericin B, although three of them, one type isolated from a neonate and two types isolated from the mother, were resistant to fluconazole. The diversity of infective strains remains alarming and encourages the consideration of several colonies per isolate or several isolates, when it is possible, per infection case. This study also points out the need to survey the susceptibility of infective strains, since some of them appear soon to be resistant to fluconazole.

Variations in the antigenic effects of tunicamycin on Candida albicans.

We report on the effect of subinhibitory doses of tunicamycin on Candida albicans cells (BP strain high responder NCYC 1466) in a defined medium favourable for expression of the mycelial phase. Tunicamycin inhibited the synthesis of some protein fractions ranging from 40 to 65 kDa, where the immunodominant antigens of C. albicans responsible for the antibody response to systemic mycosis were inhibited. By two-dimensional immunoelectrophoresis, antigen extracts from the cell cultures grown with tunicamycin showed a migration modification and a lower number of precipitation arcs with variation in their height and range.

Effect of medium composition on static and cidal activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against Aspergillus fumigatus: a multicenter study.

The effect of the medium composition on the fungistatic (MIC) and fungicidal (MLC) activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against four Aspergillus fumigatus strains has been investigated by four European laboratories. MICs were determined by broth microdilution, using RPMI 1640 and Antibiotic Medium 3 (AM3), three times in three independent determinations by the four laboratories. MLCs were determined for the three independent determinations by the four laboratories, subculturing 100 microl from each well showing no visible growth after 48 hours. Except for a 2-dilution difference observed in three cases, no differences were observed between MICs determined on the two media. In contrast, a 3- to 6-dilution discrepancy between the MLCs was observed for the azoles. Endpoints on RPMI were higher than those on AM3. A 1-2 dilution difference was noted between both the endpoints of amphotericin B and of terbinafine. The highest inter- and intra-laboratory agreements were reached on AM3. The azoles showed a medium-dependent fungicidal activity.

In vitro susceptibility of 115 isolates of Candida to amphotericin B, fluconazole and itraconazole.

Opportunistic fungal infections are an increasingly important cause of morbidity and mortality particularly due to Candida species (1). There is also an increase of candidosis especially ascribed to acquired or induced immunodeficiency syndromes or in the event of long-term antibiotic, immuno-suppressor or cytotoxic therapies. Consequently there has been an increase in the use of systemic antifungal agents responsible for the emergence of new opportunistic fungi (2) and resistant species (3, 4). Oropharyngeal candidiasis caused by various species of Candida is one of the most common opportunistic infections in AIDS. In recent years there has been an increasing number of yeast isolates resistant to fluconazole (4, 5) or to amphotericin B (6). The aim of the present study was to examine the susceptibility in vitro of itraconazole, a newly introduced antifungal agent in the local or systemic therapy of oropharyngeal candidiosis, vs well-known agents such as amphotericin B and fluconazole, against various Candida clinical isolates. The present results, in agreement with other studies, show strong in vitro activity of itraconazole against Candida spp. and particularly against less susceptible species C. glabrata, C. tropicalis or C. krusei.

Susceptibility of several species of Candida and Torulopsis to fluconazole and ketoconazole.

The authors compared the in vitro antifungal activity of fluconazole, a new triazole antifungal agent, and ketoconazole, an imidazole derivative. The MIC values were determined against 50 strains of Candida albicans, 10 strains of C. guilliermondii, 10 strains of C. krusei, 10 strains of C. parapsilosis, 10 strains of C. pseudotropicalis, 10 strains of C. tropicalis and 15 strains of Torulopsis glabrata. The fungistatic activity was evaluated by the agar dilution method using BHI and casitone media after incubation for 48 h at 28-30 degrees C. Both antifungal agents showed higher activity when tested on casitone medium; however, the G-MIC values for ketoconazole were lower than those for fluconazole.

In vitro antiplasmodial activity of stem and root extracts of Nauclea latifolia S.M. (Rubiaceae).

Aqueous extracts from Nauclea latifolia S.M. (Rubiaceae), a plant commonly used in Ivory Coast by traditional healers for the treatment of malaria, were tested on two strains of Plasmodium faliparum: FcB1-Colombia (chloroquine-resistant) and a Nigerian strain (chloroquine-sensitive). The extracts were obtained from stems and roots of the plant in two forms, infusion and decoction, both methods used by most traditional healers. The in vitro activity of N. latifolia extracts on P. falciparum was assessed both visually and by a radioactive method. The visual analysis allowed determination of the time of extract action on the erythrocytic cycle, as well as the parasitic stage of most inhibitory effect. Similar results were obtained applying fresh, frozen or lyophilized extracts. The IC50 values determined were within the range already reported for other antimalarial plants such as Azadirachta indica A. Juss (Meliaceae) or Artemisia annua L. (Asteraceae). Aqueous extracts of N. latifolia inhibited P. falciparum (FcB1 strain) mainly at the end of the erythrocytic cycle (32nd to 48th hour).

Novel anti-digoxin monoclonal antibodies with different binding specificities for digoxin metabolites and other glycosides.

Spleen cells of BALB/c mice immunized with a digoxin-bovine serum albumin conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Seven monoclonal antibodies (MAb) selected by indirect ELISA were produced, purified and characterized. All the MAb were IgG1 isotypes. The apparent equilibrium association constants (Ka) of four of the MAb, determined by Scatchard analysis of the RIA data, ranged from 1 x 10(9) M-1 to 5.9 x 10(9) M-1. The estimated Ka values of the three other MAb were found to be between 4.8 x 10(7) M-1 and 5.9 x 10(8) M-1. Using digoxin and eighteen structurally-related compounds, the seven MAb could be divided into five groups based on their binding specificities assessed by an inhibition immunoenzymatic test. The MAb in Groups I and II, in particular, showed very different specificity profiles: the two MAb in Group I had low cross-reactivity with cardioinactive digoxin metabolites, whereas the high affinity MAb in Group II recognized all the digoxin metabolites tested. The MAb in Group I might be useful in a digoxin immunoassay and the Group II MAb in therapeutic reversal of digoxin intoxication.

Production and characterization of highly specific anti-methotrexate monoclonal antibodies.

Spleen cells of a Biozzi HR mouse immunized with a bovine serum albumin-methotrexate conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty-three monoclonal antibodies (MAbs), selected by indirect ELISA, were produced and partially characterized. Using methotrexate (MTX) and eight structurally related compounds, binding specificities of the MAbs were assessed by inhibition enzyme immunoassay. All the MAbs had very low affinity for folic acid and its analogs and for the major MTX metabolite 7-hydroxymethotrexate. Using a computer cluster analysis program based on the binding specificities, the MAbs were divided into three groups. The thirteen MAbs in group I recognized primarily the pteridine portion of the MTX molecule; the eight group II MAbs recognized the benzene ring as well as the pteridine structure. The two MAbs in group III poorly distinguished between the different parts of the MTX molecule. The apparent equilibrium association constants of the anti-MTX MAbs in groups I, II, and III ranged from 7 x 10(9) to 3 x 10(8) M-1 (except for 1 MAb), from 5 x 10(7) to 6 x 10(6) M-1 (except for 2 MAbs), and from 1 x 10(6) to 3.5 x 10(5) M-1, respectively.

Production and characterization of monoclonal antibodies against human thyroglobulin.

Spleen cells of Biozzi-HR mice immunized with human thyroglobulin (hTg) were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty monoclonal antibodies (MAbs) selected by an enzyme immunoassay (indirect ELISA) were produced, purified and characterized. The equilibrium association constant (Ka) of one of the MAbs, determined by Scatchard analysis of the ELISA data, was found to be 2 X 10(9) M-1; the Ka of the other MAb, estimated from titration curves by comparison with the aforementioned MAb, ranged from 8 X 10(9) M-1 to 6 X 10(7) M-1. The reaction between the MAb and hTg was not inhibited by thyroxin (T4), triiodothyronine (T3) and triiodothyropropionic acid (DT3). Species specificity of the MAb was studied using bovine and porcine Tgb. The topology of the MAb was investigated by competitive inhibition immunoassays. Seven distinct antigenic regions were identified.