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The antimicrobial activity of Berberis heterophylla leaves, stems and root aqueous extracts was studied in vitro on Gram-positive and Gram-negative bacteria and fungi. The in vitro antifungal activity of berberine isolated from the same source against different Candida species was also investigated.
We report the first case of maxillary sinusitis caused by Actinomucor elegans in an 11-year-old patient. Histopathological and mycological examinations of surgical maxillary sinuses samples showed coenocytic hyphae characteristic of mucoraceous fungi. The fungi recovered had stolons and rhizoids, nonapophyseal and globose sporangia, and whorled branched sporangiophores and was identified as A. elegans. After surgical cleaning and chemotherapy with amphotericin B administered intravenously and by irrigation, the patient became asymptomatic and the mycological study results were negative.
Infection with Cryptococcus neoformans is an increasing problem in immunocompromised patients, particularly those with acquired immune deficiency syndrome (AIDS). Amphotericin B and fluconazole are currently acceptable therapies for cryptococcal meningitis; however, their effects remain suboptimal and recurrence or treatment failure is still a problem. Antifungal susceptibility testing may be an important tool for guiding therapy, but for C. neoformans, a reliable method is still not available. This retrospective study evaluated minimal inhibitory concentration (MIC) for amphotericin B and fluconazole, and minimal fungicidal concentration (MFC) and timed-kill curves for amphotericin B against 16 clinical isolates of C. neoformans obtained from AIDS patients with cryptococcal meningitis. No correlation between clinical outcome and MIC was observed for amphotericin B. In selected cases, the MFC seemed to be a better predictor of outcome than MIC. In this study, amphotericin B timed-kill curves appeared to show a correlation with clinical outcome of the 16 patients with AIDS-associated cryptococcal meningitis. These in vitro tests must be further evaluated in prospective studies to confirm their potential usefulness for guiding cryptococcal meningitis therapy.
The in vitro activities of three antifungal drugs alone and in combination were evaluated against five isolates of Cryptococcus neoformans using time-kill curves (TKC). The isolates were from AIDS patients who had either died or had failed to show a clinical response during amphotericin B (AMB) treatment. AMB, fluconazole (FCZ) and flucytosine (5FC), and combinations of the drugs (AMB plus 5FC, AMB plus rifampicin (RIF) and FCZ plus 5FC), were evaluated. With all five isolates AMB did not show fungicidal activity; instead, a persistent or tolerant effect was observed. Combinations of AMB plus 5FC and AMB plus RIF showed a clear synergic effect, except for one isolate tested with AMB plus RIF. In contrast, the FCZ plus 5FC combination did not inhibit growth of any isolate.
Cryptococcus neoformans is a nonfermentative yeast that requires oxygen for growth. The shaking of culture media achieves good oxygenation, promoting the growth of cryptococci. In this study, three test media (RPMI 1640, RPMI 1640-2% glucose, and buffered yeast nitrogen base ¿BYNB) recommended in the National Committee for Clinical Laboratory Standards M27A standard were examined. Growth abilities and minimum inhibitory concentrations (MICs) in microplates incubated at 35 degrees C for 48 h were determined. The results indicated that shaking and an inoculum size of 10(5) CFU/ml yielded optimal growth of this yeast. Compared to RPMI 1640, supplementation of RPMI 1640 with 2% glucose did not significantly improve growth of C. neoformans and resulted in an 8.7-h delay of exponential growth. Cryptococcal growth in RPMI 1640 at 24 h was notably better than that in RPMI-2% glucose, although by 48 h the growths were comparable. The MIC range of amphotericin B observed for the C. neoformans strains grown in RPMI 1640 with or without glucose was too narrow to allow the separation of susceptible and resistant strains based on clinical outcome. The widest ranges of MICs of flucytosine and fluconazole were obtained with BYNB. This work demonstrates the need for a new antifungal susceptibility test for C. neoformans.
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Candida dubliniensis is a recently identified chlamydospore-positive yeast species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected (HIV+) patients and is closely related to Candida albicans. Several recent reports have described atypical oral Candida isolates with phenotypic and genetic properties similar to those of C. dubliniensis. In this study 10 atypical chlamydospore-positive oral isolates from HIV+ patients in Switzerland, the United Kingdom, and Argentina and 1 isolate from an HIV-negative Irish subject were compared to reference strains of C. albicans and Candida stellatoidea and reference strains of C. dubliniensis recovered from Irish and Australian HIV+ individuals. All 11 isolates were phenotypically and genetically similar to and phylogenetically identical to C. dubliniensis. These findings demonstrate that the geographical distribution of C. dubliniensis is widespread, and it is likely that it is a significant constituent of the normal oral flora with the potential to cause oral candidiasis, particularly in immunocompromised patients.
A rapid method to evidence urease activity is described. Urea hydrolysis and consequent production ammonia are detected by a chemical reaction producing a blue phenol compound (indophenol blue). Three hundred and three yeast were tested. Out of 107 urease-positive organisms detected by Christensen's Urea Agar Test (CUAT) 102 were positive by our method. No false negatives were observed by this method when testing 87 Cryptococcus strains. Ths practical screening test for presumptive identification of Cryptococcus neoformans is simple, unaffected by pH changes and requires 15 minutes to be performed.
In 1992, the NCCLS proposed a broth macrodilution method for determining the in vitro susceptibility of yeast. However, for dimorphic fungi no standardised method is available. The aim of our study was to evaluate the reference broth macrodilution method with isolates of Paracoccidioides brasiliensis. The in vitro susceptibility of two ATCC strains (32069 and 36324) and nine clinical isolates were determined against serial dilutions of amphotericin B (AMB), ketoconazole (KTZ), and itraconazole (ITZ) from 0.02 to 20 mg/l and fluconazole (FCZ) from 0.1 to 100 mg/l. The inoculum used was 0.5 x 10(4)-2.5 x 10(5) CFU/ml, employing the yeast phase in order to obtain homogeneous growth. The test was incubated with agitation in a rotating shaker at 35 degrees C, and 7 days was the optimal time for MIC final reading. Average MIC50/MIC90 (mg/l) for the eleven isolates tested were the following: AMB: < 0.02/0.16, FCZ: 1.6/6.2, KTZ: 0.08/0.31, ITZ: 0.31/1.3. One clinical isolate showed high MIC for azole drugs (KTZ: 1.3 mg/ml, FCZ: 100 mg/l and ITZ 5 mg/l). Although further studies are necessary to evaluate the usefulness of this methodology for P. brasiliensis, the latter may be clinically useful to predict the development of resistance.
A multicenter study was performed between April and September 1998 with the participation of 12 medical centers located in 8 different provinces and in the capital city of Argentina. The aim of this study was to determine the species distribution and the antifungal susceptibility profile of isolates causing nosocomial fungal infections. All the fungal isolates obtained were sent to the Mycology Department for reference identification and antifungal susceptibility testing. Eighty-nine isolates were received from different clinical specimens. The distribution of species obtained was C. albicans (50.6%), C. tropicalis (22.5%), C. parapsilosis (20.2%), C. krusei (3.4%), C. glabrata (2.2%) and Debaryomyces hansenii (1.1%). Most of the isolates (85/88) had MICs for amphotericin B < or = 1 microgram/ml, C. krusei showed resistance to fluconazole but was dose dependent susceptible to itraconazole, C. glabrata (2/2) were resistant against both drugs, most of the isolates of C. albicans, C. tropicalis and C. parapsilosis were susceptible to these triazole drugs. These data showed a different distribution of Candida species compared with results obtained in other countries. The low frequency of appearance of C. krusei and C. glabrata in our country suggests a reduced selective pressure by triazoles.
Dermatomycoses are an important sanitary problem. Our country counts with partial epidemiological data only referred to a few geographical areas. The aim of our work was to perform a multicenter study with the participation of 15 medical centers located at the Capital City and in 11 Provinces of Argentina with different socioeconomical and cultural characteristics. Between October and December 1998, 851 ambulatory patients of both sexes (58% female, 42% male) with lesions compatible with mycoses in skin, nails or scalp were studied. Only 51.6% of the patients had mycoses confirmed by direct microscopic examination and/ or culture. Dermatophytes caused 69.3% of the diagnosticated infections, yeast 12.2% and Malassezia furfur 13.3%. Agents of dermatomycoses most frequently isolated were T. rubrum (52.7%), T. mentagrophytes (19.2%) and M. canis (14.2%). C. albicans was the predominant causative agent of yeast infections (44.4%). In children below 11 years old, the most frequent infections were tinea capitis 55.4% (M. canis 60%, T. tonsurans and M. gypseum 8.3%) and tinea corporis 20% (M. canis 46%, T. mentagrophytes 30%). The most frequent localizations in adult were toenail and feet (63%) (T. rubrum 47.3% and 45.7%, T. mentagrophytes 9.5% and 23.4%, respectively), fingernail 15.6% (C. albicans 25% and T. rubrum 8.3%) and trunk and extremities 12.2% (M. furfur 57.5% and T. mentagrophytes 14.9%). Direct examination allowed the diagnosis of 94.66% of all positive samples and 91.74% of the cases with positive isolation.
We analyzed the in vitro infection process by P. brasiliensis and the effect of extracellular factor(s) produced on monolayers of mammalian Vero cell lines. The yeast phase of four strains was studied: B339 (avirulent or slightly virulent), U, (intermediate virulence), 93745 and 63265 (both highly virulent). Strains of intermediate and high virulence had higher adherence at first contact (about 16%). Strain B339 had a slower adherence at first contact (8%) than the others during the same period. The production of extracellular proteases, soluble extracellular factor(s) and extracellular antigen gP43 showed no correlation with the in vitro physiopathogenicity of the analyzed strains. We demonstrate that the Vero model presented in this paper is a suitable system to study infection and virulence in vitro. We are currently assessing its usefulness as a tool for the analysis of the interaction between pathogen, host and antifungal agents.
Candida spp. colonization in neonates occurs due to vertical or horizontal transmission. Preliminary studies determined that Candida albicans is the principal agent of these infections. In order to establish nosocomial transmission, 26 Candida albicans strains isolated from patients with candidosis hospitalized during a 18-month period in 2 neonatal intensive care units (NICU) from a pediatric hospital were studied. Fourteen isolates from patients and health care workers, involved in possible outbreaks of an intensive care unit (UCI) and a NICU from another pediatric hospital were also studied. All Candida albicans strains were genotyped by Southern blot hybridization with 27A. Isolates for outbreak confirmation were also hybridized with another specific Candida albicans probe, Ca3. Hybridization patterns demonstrated horizontal transmission in all the units studied. In a NICU, transmission among 4 patients during a 10-month period could be established and in the other NICU, 3 cases of transmission among 2 patients each were demonstrated in periods of 2 to 20 days. The outbreak studies showed the same strain isolated from 2 nurses and from one patient at the NICU and at the ICU identical strains were found in 3 patients. In this study, hybridization with Ca3 in addition to 27A probe did not increase discrimination power among isolates. Genotypic analysis allows, not only, determination of transmission and persistence of strains during prolonged periods or in sporadic outbreaks, but also facilitates necessary epidemiological decisions for optimizing nosocomial fungal infection control measures.
The aim of our work was to determine the genotypic relatedness among H. capsulatum isolates from Argentinian patients with or without AIDS and isolates from other sources and geographic origins. We analyzed band patterns of 16 isolates obtained by RAPD-PCR, 11 Argentinian clinical isolates (8 from patients with AIDS and 3 from patients without AIDS), 2 strains from the CDC, 2 isolates from Mexico and one isolate from Uruguay. With the band patterns obtained, we built a dendrogram, which showed 3 major groups. Group I contained 2 isolates from Mexico, group II 1 strain from CDC and group III included 13 isolates, and was divided into 4 subgroups. Subgroup IIIa contained 2 isolates from patients with and without AIDS. Subgroup IIIb included isolates from patients without AIDS from Chaco. Subgroup IIIc contained 7 isolates from Argentina (from Buenos Aires province) and the Uruguayan isolate (6 isolates from patients with AIDS). Subgroup IIId included only one strain from CDC. All the Argentinian and the Uruguayan isolates went into group III, subgroups IIIa, IIIb and IIIc. This is a preliminary study; however, a relatedness of the RAPD-PCR band patterns and the geographic origin was observed. Although there might be a probable association between immunocompromised patients with AIDS and infective capability of some H. capsulatum isolates, further studies with a larger number of isolates from different sources and geographic origins are necessary to confirm these results.
Candida vaginitis is one of the most frequent infection of the female genital tract with a high incidence. Approximately 75% of sexually active women suffer at least one episode of Candida vaginitis and 10% of them have recurrent episodes. Pregnancy, diabetes mellitus and antibiotic treatment are the most common predisposing factors, C. albicans is the etiologic agent most frequently found. The widespread reports of fluconazole resistance in Candida species and the selection of non Candida albicans prompted the study of species distribution of vulvovaginal candidiasis and their in vitro susceptibility against current antifungal agents. A total of 314 women with vaginal infection were studied. Yeasts were isolated from 104 patients with vulvovaginal candidiasis. The following species were identified: C. albicans 87.5%, C. glabrata 8.6% and 3.9% included C. krusei, C. famata, C. tropicalis and S. cerevisiae. The minimal inhibitory concentration (MIC) was determined for nystatin, isoconazole, fluconazole and ketoconazole, using a broth microdilution method based on NCCLS procedure. Although most of the isolates were C. albicans, the high percentage of C. glabrata recovered suggests the need to identify the yeasts isolated. Fluconazole resistant C. albicans were isolated in 13.46% of the cases. Thus, further studies are required to correlate the possible role of these strains in recurrent vulvovaginal candidiasis.
During the past decades, several studies have reported an increase in the incidence of nosocomial candidosis. In a prospective study, performed at the Departamento de Micología, INEI, ANLIS Dr. C. G. Malbrán and the Servicio de Neonatología and Microbiología, Hospital de Niños Sor María Ludovica, from October 1995 to December 1996, 167 patients with candidosis were detected. Candida species isolated were C. albicans (53.1%), C. parapsilosis (26.5%) and C. tropicalis (14.8%). The aim of this work was to characterize the clinical C. parapsilosis isolates from pediatric patients hospitalized in two neonatal intensive care units from the same hospital and to evaluate the usefulness of electrophoretic karyotype (EK) and restriction endonuclease analysis of genomic DNA (REAG) using a low frequency digestion enzyme. EK of all isolates disclosed 12 banding patterns and REAG with endonuclease Sfi I showed only 5 groups. However, isolates from the control group could not be separated from the clinical isolates. The isolates within each dendogram group for EK or REAG were apparently unrelated. Our results show that EK yields better results than REAG, but that it falls short of the desired discrimination, which suggests that these techniques do not seem to be useful for studying nosocomial C. parapsilosis outbreaks.
C. neoformans (Cn) causes severe meningitis in immunocompromised patients, particularly those with AIDS. Little is known about Cn serotypes associated with human infections in Argentina due to the lack of antisera availability. The aim of this study was to produce these antisera, to serotype the Argentinean clinical isolates of Cn received at our laboratory from 1984 to 2001 and to evaluate the agreement of these results with the variety differentiation obtained with Canavanine-Glycine-Bromothymol Blue agar (CGB) medium. We studied 123 isolates recovered from first episodes of cryptococcosis from HIV-infected patients (85), non HIV-infected patients (6) and from other non specified patients (32). Of the isolates, 89% (110/123) were serotype A, 4% (5/123) corresponded to serotype D, 3% (3/129) were serotype AD, 3 isolates were untypable and 2 corresponded to serotype B. All the serotyped isolates agreed with the variety determined by CGB medium. These results indicate that, in our country, most cryptococcal infections in patients with AIDS are associated with serotype A (77/85), which agrees with international studies. In patients with other immunosuppressions, this serotype may be also predominant, although a low number of isolates were tested (4/6). Recovery of Cn serotype B in these clinical isolates suggests that studying the variety and their serotypes might be important to detect a probable epidemiological alteration.
Two strains of C. neoformans var. gattii serotype B were isolated from a park in Buenos Aires city, Argentina. Samples were collected in spring, rubbing with swabs on the inner hollow of Eucalyptus spp. and other trees, which were not identified at the collection moment. Samples were swabbed directly onto cafeic acid with antibiotic agar, then incubated at 28 degrees C and observed daily for a week. Isolates were recovered from cafeic acid medium showing characteristic colonies of the species that allowed differentiation from other microorganisms of the sample. Identification was performed using carbohydrate fermentation, assimilation of nitrogen and carbon sources, and urease and phenoloxidase detection. Strain variety was determined with canavanine-glicine-bromotimol blue agar medium (CGB). Genotypic characterization using AP-PCR with (GACA)4 aleatory primer showed that both band profiles could be differentiated by only one band. These two strains were isolated from tree species different from Eucalyptus spp. These isolates from natural sources demonstrate that C. neoformans var. gattii efosts in Buenos Aires city environment and that it is not necessarily associated with Eucalyptus spp. The presence of C. neoformansvar. gattii in the environment, detected for the first time in Argentina, may explain its sporadic association with human infections in the local population.