In vitro activity of the lipopeptide derivative (Pal-Lys-Lys-NH), alone and in combination with antifungal agents, against clinical isolates of dermatophytes.

BACKGROUND: An increasing number of antimycotics have become available for the treatment of dermatophytoses; however, there are reports suggesting recalcitrance to therapy or resistance of a dermatophyte against conventional treatment. Lipopeptides represent novel therapeutic drugs with a new mode of action. OBJECTIVES: The aim of this study was to investigate the in vitro effects of the lipopeptide Pal-Lys-Lys-NH(2) (PAL) alone and in combination with standard antifungal agents, such as fluconazole (FLU), itraconazole (ITRA) and terbinafine (TER) against 24 clinical isolates of dermatophytes belonging to four species. METHODS: A broth microdilution method following the Clinical and Laboratory Standards Institute recommendations (M38-A) was used for testing drugs alone and in combination. RESULTS: PAL minimum inhibitory concentrations (MICs) ranged from < or = 0.25 to > 16 microg mL(-1) and they were similar to those of FLU and higher than those of either ITRA or TER. Synergy, defined as a fractional inhibitory concentration (FIC) index of < or = 0.50, was observed in 67%, 52% and 15% of PAL/ITRA, PAL/TER and PAL/FLU interactions, respectively. None of these combinations yielded antagonistic interactions (FIC index > 4). When synergy was not achieved, there was still a decrease in the MIC of one or both drugs used in the combination. CONCLUSIONS: Our study demonstrates that PAL has potential activity against dermatophytes. In addition, the in vitro activity of PAL can be enhanced upon combination with standard drugs. This lipopeptide applied in the form of lacquer, spray or ointment, could represent an interesting new therapy, particularly when combined with conventional treatment in recalcitrant or resistant dermatophyte infections.

Fluconazole susceptibility of Candida spp. isolates collected over nine years in a teaching hospital of Ancona, Italy.

The in vitro activity of fluconazole was investigated against 476 yeast isolates collected during a 9-year period (1997-2005) from patients hospitalised in a teaching hospital of Ancona. They included 373 isolates of Candida albicans, 53 of Candida glabrata and 50 of Candida parapsilosis. Minimum inhibitory concentrations (MICs) determined in accordance with the Clinical Laboratory Standards Institute methodology showed that 96% of the isolates were susceptible (MIC < or =8.0 microg/ml). The uncommon, resistant isolates (MIC > or =64 microg/ml) were randomly distributed over time. Our data show that resistance to fluconazole in this geographical area is a rare event and suggest that this triazole can still represent first-line therapy in our institution.

In-vitro activity of the synthetic protegrin IB-367 alone and in combination with antifungal agents against clinical isolates of Candida spp.

The in vitro activity of the peptide IB-367, alone or combined with either fluconazole (FLU) or amphotericin B (AMB), was investigated against 25 Candida isolates belonging to five species. IB-367 minimum inhibitory concentrations (MICs) ranged from 2.0 to 32 microg/ml and it was active against both FLU-susceptible and - resistant isolates. A rapid fungicidal activity was observed. Synergism was documented in 41.6% and 44% of IB-367/FLU and IB-367/AMB interactions, respectively. Antagonism was never observed. The broad antifungal activity and the positive interactions with AMB and FLU suggest that IB-367 represents a promising candidate against infections due to Candida spp.

Genotypic variation and antifungal susceptibilities of Candida pelliculosa clinical isolates.

At the Istituto Ricovero Cura Carattere Scientifico, Ospedale Maggiore di Milano, Italy, Candida pelliculosa accounted for 3.3 and 4.4 % of all Candida species other than Candida albicans collected during 1996 and 1998, respectively. Genetic variability was investigated by electrophoretic karyotyping and inter-repeat PCR, and the susceptibility to five antifungal agents of 46 strains isolated from 37 patients during these 2 years was determined. Combination of the two typing methods yielded 14 different DNA types. Although the majority of DNA types were randomly distributed among different units, one DNA type was significantly more common in patients hospitalized in a given unit compared with those from other wards (P=0.034), whereas another DNA type was more frequently isolated in patients hospitalized during 1996 than in those hospitalized during 1998 (P=0.002). Fluconazole, itraconazole and posaconazole MIC90 values were 16, 1 and 4 microg ml-1, respectively. All isolates but three were susceptible in vitro to flucytosine. All isolates were susceptible in vitro to amphotericin B. These data suggest that there are possible relationships among strains of C. pelliculosa, wards and time of isolation. Amphotericin B seems to be the optimal drug therapy in infections due to this yeast species.

Comparative analysis of pathogenicity of Cryptococcus neoformans serotypes A, D and AD in murine cryptococcosis.

OBJECTIVES: To characterize the pathogenicity of 15 strains of Cryptococcus neoformans belonging to several serotype/mating type allele patterns (Dalpha, Da, A(alpha), A(a), A(alpha)/D(a) and D(alpha)/A(a)) in experimental models of murine cryptococcosis. METHODS: CD1-infected mice were examined for survival and fungal loads in either brain or lung during the course of infection. RESULTS: All strains, with the exception of one Da strain, produced melanin in vitro. Similarly, all strains were encapsulated and produced phospholipase. When CD1 mice were challenged intravenously (i.v.) with 5x10(5)CFU/mouse and observed for 60 days post-infection, a significant variation of mortality rate was observed among mice infected with different strains. A(alpha) and A(alpha)/D(a) strains all produced 100% mortality within the study period with mean survivals significantly shorter than those of mice infected with strains belonging to any other allele type (P<0.0001). A wide range of pathogenicity was shown by haploid and diploid strains presenting D(alpha) allele. This finding was confirmed by an intranasal model of challenge. To investigate the progression of infection, the mice were challenged i.v. with 5x10(4)CFU/mouse and tissue burden experiments (brain and lung) were performed on days 6 and 12 post-infection. Only the mice infected with A(alpha) and A(alpha)/D(a) strains showed a >1 log(10) increase of CFU/g in both tissues throughout the study period. CONCLUSIONS: Our results suggest that the presence of the A(alpha) mating type allele in either haploid or diploid strains is correlated with virulence, while the presence of the A(a) or D(a) allele in haploid strains is associated with moderate or no virulence. Finally, either haploid or diploid strains presenting D(alpha) allele vary in virulence.

When to start highly active antiretroviral therapy in chronically HIV-infected patients: evidence from the ICONA study.

OBJECTIVES: To compare the response to highly active antiretroviral therapy (HAART) in individuals starting HAART at different CD4 cell counts. DESIGN: The mean increase in CD4 cell count and rate of virological failure after commencing HAART were measured in antiretroviral-naive patients (1421) in a large, non-randomized multicentre, observational study in Italy (ICONA). Clinical endpoints were also evaluated in a subset of patients who started HAART with a very low CD4 cell count. RESULTS: After 96 weeks of therapy, the mean rise in CD4 cell count was 280, 281 and 186 x 10(6) cells/l in patients starting HAART with a CD4 cell count < 200, 201--350 and > 350 x 10(6) cells/l, respectively. Patients starting HAART with a CD4 cell count < 200 x 10(6) cells/l tended to have a higher risk of subsequent virological failure [relative hazard (RH), 1.15; 95% confidence interval (CI), 0.93--1.42] compared with patients starting with > 350 x 10(6) cells/l. There was no difference in risk between the 201--350 and the > 350 x 10(6) cells/l groups (RH, 1.0; 95% CI, 0.79--1.29). The incidence of new AIDS-defining diseases/death in patients who started HAART with a CD4 count < 50 was 0.03/person-year (95% CI, 0.10--0.33) during the time in which the patient's CD4 cell count had been raised to > 200 x 10(6) cells/l. CONCLUSIONS: There was no clear immunological or virological advantage in starting HAART at a CD4 cell count > 350 rather than at 200--350 x 10(6) cells/l. The increase in CD4 cells restored by HAART is meaningful in that they are associated with reduced risk of disease/death.

A randomized trial comparing the introduction of ritonavir or indinavir in 1251 nucleoside-experienced patients with advanced HIV infection.

ISS-IP1, a multicenter, randomized, 48-week open trial, was designed to compare the introduction of ritonavir or indinavir in patients with previous nucleoside experience and CD4+ cell counts below 50/mm3. Concomitant antiretroviral treatment with nucleoside analogs was allowed. Primary efficacy measures were survival and time to a new AIDS-defining event or death, analyzed through the whole period of observation by the intention-to-treat approach. Primary toxicity measures were time to treatment discontinuation and adverse events, grade at least 3/serious, analyzed by an on-treatment approach. Evaluation-of efficacy also included CD4+ cell and RNA response. The trial enrolled 1251 patients in 5 months. At baseline, mean CD4+ cell count was about 20 cells/mm3 and mean HIV RNA copy number was 4.9 log10/ml in both groups. Overall, 402 patients in the ritonavir group and 250 patients in the indinavir group permanently discontinued the assigned treatment (relative risk, 1.96; 95% CI, 1.68-2.30; p = 0.0001), with most of this difference dependent on a higher number of discontinuation for adverse events in the ritonavir group. After a mean follow-up of 307 days (ritonavir, 304; indinavir, 309), 124 deaths (ritonavir, 61; indinavir, 63; relative risk, 0.96; 95% CI, 0.67-1.36; p = 0.80) and 330 new AIDS-defining events (ritonavir, 170; indinavir, 160; relative risk, 1.05; 95% CI, 0.85-1.31; p = 0.60) were observed. CD4+ cell counts increased in both groups in patients still receiving treatment, with about 100 cells gained by week 24 and 150 cells gained by week 48. Body weight also increased over time in both groups. Analysis of RNA response showed a decrease of 1.5 log10 or higher in both treatment groups. Overall, 400 patients in the ritonavir group and 338 patients in the indinavir group developed at least one grade 3/serious new adverse event during follow-up (relative risk, 1.48; 95% CI, 1.28-1.72; p = 0.0001). Favorable CD4+ cell and RNA responses at 24 and 48 weeks were observed in both groups of patients remaining on treatment. Indinavir showed slightly better effects in sustaining RNA, CD4+ cell, and body weight responses. Ritonavir and indinavir results were comparable in terms of clinical outcome (survival and AIDS-defining events).

Electron microscope study of a case of Australia antigen-positive chronic hepatitis.

The laboratory findings in a case of Australia antigen (Au-Ag) positive chronic hepatitis in a 4-year-old boy are given. Studies by the electron microscope technique of thin sectioning revealed the presence of 200A particles in the nuclei of hepatocytes. These particles are identical to those described previously in occasional specimens of Au-Ag-positive liver tissue. However, the pattern found here differs from previous descriptions inasmuch as no heavily infected nuclei could be found but almost all nuclei could be shown to contain extremely small numbers of these particles. The significance of this finding is discussed and the danger that it would be easy to obtain false negatives in similar tissue pointed out.

In-vitro activity and killing effect of polycationic peptides on methicillin-resistant Staphylococcus aureus and interactions with clinically used antibiotics.

The in-vitro activity of nisin, a 34-residue peptide produced by several Lactococcus lactis strains, and ranalexin, a 20-residue peptide isolated from the skin of the bullfrog Rana catesbeiana, alone and in combination with amoxycillin, amoxycillin-clavulanate, imipenem, clarithromycin, ciprofloxacin, rifampin and vancomycin was investigated against 40 nosocomial isolates of methicillin-resistant Staphylococcus aureus (MRSA). All isolates were inhibited at concentrations of 1 to 32 microg/ml. Synergy was observed when the peptides were combined with other agents, with the exception of the beta-lactams. Finally, the consecutive exposures to each peptide did not result in selection of stable mutants with decreased susceptibility. Our finding show that nisin and ranalexin are active against MRSA, and that their activity is enhanced when they are combined with several antimicrobial agents.

Outbreak of fungemia due to Candida parapsilosis in a pediatric oncology unit.

We report an outbreak of infection due to genotypically identical Candida parapsilosis isolates among patients hospitalized in a pediatric oncology unit. Control cultures showed genetic relatedness between strains isolated from the patients and those isolated from the hands of a health care worker. Our data underline the importance of an effective surveillance program for preventing nosocomial fungal infections.

Electrophoretic karyotype and in vitro antifungal susceptibility of Cryptococcus neoformans isolates from AIDS patients.

Electrophoretic karyotype (EK) was used to type 13 clinical isolates of Cryptococcus neoformans from eight AIDS patients. All of the isolates were also tested for their in vitro susceptibilities to fluconazole, itraconazole, D0870, flucytosine, and amphotericin B by a broth macrodilution technique performed according to the National Committee for Clinical Laboratory Standards recommendations. Although all strains were isolated from a limited geographic area, DNA typing showed a wide genetic variation in this group of patients, yielding seven different patterns. Two patients in whom C. neoformans was isolated in the same time period shared similar EK profiles, suggesting the possibility of cross-infection. In three patients, sequential isolates were evaluated: in two of them, EK analysis showed the persistence of the same genotype throughout the infection, whereas from the third, two isolates of C. neoformans with two different DNA profiles were obtained. Despite the small number of strains considered in this study, our susceptibility data indicate that C. neoformans isolates are very susceptible to the new triazoles.

Antimicrobial activity of polycationic peptides.

The in vitro activity of six polycationic peptides, buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin, were evaluated against several clinical isolates of gram-positive and gram-negative aerobic bacteria, yeasts, Pneumocystis carinii and Cryptosporidium parvum, by using microbroth dilution methods. The peptides exhibited different antibacterial activities and rapid time-dependent killing. The gram-negative organisms were more susceptible to buforin II and cecropin P1, whereas buforin II and ranalexin were the most active compounds against the gram-positive strains. Similarly, ranalexin showed the highest activity against Candida spp., whereas magainin II exerted the highest anticryptococcal activity. Finally, the peptides showed high anti-Pneumocystis activity, whereas no compound had strong inhibitory effect on C. parvum.

Combination studies between polycationic peptides and clinically used antibiotics against Gram-positive and Gram-negative bacteria.

The in vitro interaction between five polycationic peptides, buforin II, cecropin P1, indolicidin, magainin II, and ranalexin, and several clinically used antimicrobial agents was evaluated against several clinical isolates of Gram-positive and Gram-negative aerobic bacteria, using the microbroth dilution method. The combination studies showed synergy between ranalexin and polymyxin E, doxycycline and clarithromycin. In addition, magainin II was shown to be synergic with betalactam antibiotics.

Analysis of antibody response to Cryptococcus neoformans in five patients with AIDS and cryptococcosis by immunoblotting.

OBJECTIVES: The serological response of five patients with AIDS and cryptococcosis to non capsular antigens from Cryptococcus neoformans var. neoformans has been investigated. METHODS: Pressates of different isolates of C. neoformans were used as antigenic preparation for immunoblotting of patient samples. RESULTS: Multiple sera and cerebrospinal fluids sequentially collected from five AIDS patients with cryptococcosis showed a wide heterogeneity in antibody response with bands at 48. 43, 38 and 26 kD being present in all clinical samples of all five patients. The variation in banding patterns of the sequential samples from three patients was correlated with a decrease of the antigen titre and with an amelioration of the cryptococcal infection. CONCLUSIONS: We identified antibodies to four immunodominant non-capsular antigens, which might represent major target molecules of the humoral response of patients with cryptococcosis.

Infection due to Absidia corymbifera in a patient with a massive crush trauma of the foot.

We report the case of a patient with a massive crushing trauma of the right foot who developed a local infection due to Absidia corymbifera. Systemic and local antifungal therapy with ketoconazole associated with hyperbaric oxygen therapy (HBO) yielded a rapid clinical and microbiological resolution. Controlled clinical studies are warranted to further elucidate the potential utility of HBO/antifungal combination therapy.

Cyclosporiasis in a traveler returning from South America.

Cyclospora is a coccidian, previously referred to as a cyanobacterium-like body, with an epidemiology similar to that of Cryptosporidium parvum. In recent years, several studies have shown that Cyclospora is not a rare opportunistic pathogen but rather is the cause of common, worldwide intestinal infections in healthy adults and children. Previous reports of diarrhea resulting from Cyclospora sp. have been linked to travelers and immunocompromised patients. The species has a worldwide distribution. Although Cyclospora infection has been reported from Southeast Asia, Papua New Guinea, Indonesia, India, Pakistan, Nepal, the Middle East, North Africa, the United Kingdom, the Caribbean, the United States, Central America, and South America, the true prevalence of this parasite in any population is unknown. The parasite is associated with prolonged self-limiting and relapsing watery diarrhea, anorexia, fatigue, and sometimes myalgia. Fever is infrequent.

Genotyping and antifungal susceptibility of human subgingival Candida albicans isolates.

Subgingival colonization by Candida albicans has been described in human immunodeficiency virus (HIV)-infected individuals, but subgingival isolates have scarcely been characterized, particularly with respect to genotype and antifungal susceptibility. A series of 29 subgingival strains of C. albicans isolated from nine HIV-infected individuals was typed by electrophoretic karyotyping and tested for susceptibility to fluconazole, itraconazole, the new investigational triazole posaconazole and amphotericin B. DNA typing showed genetic heterogeneity within subgingival isolates, as almost every individual harbored his/her own specific isolate. Genetic identity was usually demonstrated within oral and subgingival isolates simultaneously collected from the same individual, but a number of DNA types were found to be unique to subgingival strains. These findings suggest that colonization is not just the result of Candida spreading from oral surfaces, and that subgingivally adapted strains could be involved. All isolates were susceptible to all the triazole drugs tested and amphotericin B. Additional studies on subgingival Candida colonization and further characterization of subgingival isolates are now required to clarify the role of Candida as opportunistic periodontal pathogen.

Imipenem/cilastatin treatment of acute infections in AIDS/ARC patients.

We studied the clinical efficacy and safety of imipenem, a broad spectrum beta-lactam antibiotic, in acute bacterial infections in 21 patients with AIDS or AIDS-related complex (ARC). Imipenem/cilastatin was administered as a 30-min intravenous infusion using a dose of 500 mg/8 h. Bacterial pathogens were isolated before treatment in 80% of cases; 87.5% of all strains were susceptible to imipenem in vitro. Treatment resulted in rapid control of the infections in 80% of patients. Clinical and laboratory adverse reactions probably related to imipenem treatment were noted in 8 patients.

Fluconazole, itraconazole and ketoconazole in-vitro activity. A comparative study.

We compared in-vitro activity of fluconazole, itraconazole and ketoconazole by evaluating their Minimal Inhibitory Concentrations (MICs) for 100 fungal strains isolated from different biological specimens of ARC/AIDS patients. A semisolid agar medium was used: this method is suitable for testing molds and yeasts, and can be applied to all azole antifungal drugs. Fluconazole had higher MICs than two other tested drugs, especially for Candida krusei strains; however it never had a MIC higher than 40 mg/l. Itraconazole and ketoconazole had MICs higher than 40mg/l for one Cryptococcus neoformans strain. There were no significant differences for itraconazole and ketoconazole among the tested strains.

Comparison of broth dilution and semisolid agar dilution for in vitro susceptibility testing of Cryptococcus neoformans.

We compared the in vitro activity of amphotericin B, flucytosine, itraconazole, fluconazole, ketoconazole and miconazole against 18 strains of Cryptococcus neoformans by using two methods: microbroth dilution and semisolid agar dilution. By both of the methods minimum inhibitory concentrations (MICs) showed a wide range for all antifungal agents but not for amphotericin B. Statistically significant differences between the two methods were observed only with amphotericin B and flucytosine, p = 0.048 and p = 0.045 respectively. Our study suggests that azole susceptibility testing for C. neoformans may be performed by the broth microdilution as well as the semisolid agar test. The choice of the method when testing amphotericin B and flucytosine is more problematic.