Fungal and bacterial coinfections increase mortality of severely ill COVID-19 patients.

BACKGROUND: SARS-CoV-2 predisposes patients to secondary infections; however, a better understanding of the impact of coinfections on the outcome of hospitalized COVID-19 patients is still necessary. AIM: To analyse death risk due to coinfections in COVID-19 patients. METHODS: The odds of death of 212 severely ill COVID-19 patients were evaluated, with detailed focus on the risks for each pathogen, site of infection, comorbidities and length of hospitalization. FINDINGS: The mortality rate was 50.47%. Fungal and/or bacterial isolation occurred in 89 patients, of whom 83.14% died. Coinfected patients stayed hospitalized longer and had an increased odds of dying (odds ratio (OR): 13.45; R2 = 0.31). The risk of death was increased by bacterial (OR: 11.28) and fungal (OR: 5.97) coinfections, with increased levels of creatinine, leucocytes, urea and C-reactive protein. Coinfections increased the risk of death if patients suffered from cardiovascular disease (OR: 11.53), diabetes (OR: 6.00) or obesity (OR: 5.60) in comparison with patients with these comorbidities but without pathogen isolation. The increased risk of death was detected for coagulase-negative Staphylococcus (OR: 25.39), Candida non-albicans (OR: 11.12), S. aureus (OR: 10.72), Acinetobacter spp. (OR: 6.88), Pseudomonas spp. (OR: 4.77), and C. albicans (OR: 3.97). The high-risk sites of infection were blood, tracheal aspirate, and urine. Patients with coinfection undergoing invasive mechanical ventilation were 3.8 times more likely to die than those without positive cultures. CONCLUSION: Severe COVID-19 patients with secondary coinfections required longer hospitalization and had higher risk of death. The early diagnosis of coinfections is essential to identify high-risk patients and to determine the right interventions to reduce mortality.

Cinnamyl Schiff bases: synthesis, cytotoxic effects and antifungal activity of clinical interest.

The aim of this study was to synthesize and investigate the in vitro antifungal properties of 23 cinnamyl Schiff bases. In addition, cytotoxic effects of such cinnamyl Schiff bases against human lung, kidney or red blood cells were also checked. The compounds were synthesized in a single-step, 2 min of reaction under microwave irradiation produced up to 97% yield. Six of the 23 cinnamyl Schiff bases possessed antifungal activities against strains of Candida, Aspergillus, Fonsecaea and, particularly, Cryptococcus species. Indeed, cinnamyl Schiff bases 1 and 23 exhibited minimum inhibitory concentration (MIC) values more than twofold lower than fluconazole (FCZ) against all the Cryptococcus neoformans strains (MIC = 1·33, 1·4 and 5·2 µg ml-1 , respectively) and Cryptococcus gattii strains (MIC = 5·3, 2·8 and 9·2 µg ml-1 , respectively) (12 strains of each species) while cinnamyl Schiff base 11 was as potent as FCZ against all strains from both Cryptococcus species. No significant cytotoxic effects were observed for Schiff bases against human lung, kidney or red blood cells, all presenting selective indexes higher than 10. In conclusion, this study revealed cinnamyl Schiff bases, especially 1 and 23, as new lead anticryptococcal agents for the discovery of novel antifungal drugs. SIGNIFICANCE AND IMPACT OF THE STUDY: The occurrence and severity of fungal infections have increased in recent decades due to resistance to available antifungal drugs and the appearance of new emerging pathogens. Thus, the search for new antifungal agents is mandatory. From a series of 23 cinnamyl Schiff bases, two compounds (1 and 23) were interrogated as new anticryptococcal agents without significant cytotoxicity against human lung, kidney or red blood cells. In turns, these new Schiff bases are lead compounds for the discovery of novel antifungal drugs.

Social, environmental and microbiologic aspects of endemic mycoses in Brazil.

Many factors that lead to host immunosuppression are clearly known to predispose the host to fungal diseases, significantly influencing the occurrence of mycoses. However, little or nothing has been discussed regarding social or economic factors that can influence the occurrence of diseases caused by fungi. In this minireview, we discuss several factors that may affect the occurrence of mycoses in Brazil, a continentally extended country that is marked by large climatic variations and severe socioeconomic distortions that may limit access to health services for the population.

Genomic and epidemiological monitoring of yellow fever virus transmission potential.

The yellow fever virus (YFV) epidemic in Brazil is the largest in decades. The recent discovery of YFV in Brazilian Aedes species mosquitos highlights a need to monitor the risk of reestablishment of urban YFV transmission in the Americas. We use a suite of epidemiological, spatial, and genomic approaches to characterize YFV transmission. We show that the age and sex distribution of human cases is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally reveals an early phase of sylvatic YFV transmission and spatial expansion toward previously YFV-free areas, followed by a rise in viral spillover to humans in late 2016. Our results establish a framework for monitoring YFV transmission in real time that will contribute to a global strategy to eliminate future YFV epidemics.

Changes in phase angle and body composition induced by resistance training in older women.

BACKGROUND: Resistance training (RT) has selective effects on body composition that may counteract the deleterious effects of aging. Changes in phase angle (PhA) may serve to monitor the influence of RT in older people. OBJECTIVES: To describe the effect of RT in training, detraining and retraining on body composition, including PhA in older women. SUBJECTS/METHODS: Thirty-three older women (⩾60 years old) participated. The RT program was carried out over 12 weeks for each stage (training, detraining and retraining). Whole-body fat-free mass and fat mass (FM) and appendicular lean soft tissue (ALST) measurements were carried out using a dual energy X-ray absorptiometry. Bioimpedance spectroscopy was used to estimate total body water (TBW), intra (ICF) and extracellular (ECF) fluids, whole-body resistance (R) and reactance (Xc) and PhA. Upper and lower body muscle strength were also assessed. RESULTS: From baseline to after training muscle strength, ALST and PhA significantly (P<0.05) increased. In detraining, significant (P<0.05) reductions in muscle strength, TBW, ECF, ICF and PhA along with significant (P<0.05) increases in R were observed, with the greatest magnitude observed for PhA (Δ=-7.6%). From detraining to retraining a significant reduction in FM along with increases in Xc, PhA and muscle strength were observed. Although an increase was observed from detraining to retraining in PhA, the values were still lower than baseline PhA. CONCLUSIONS: In untrained older women, a RT is associated with increases in PhA, whereas detraining results in a marked decrease in PhA, and more time may be required in retraining to counteract the negative influence of absence of exercise stimulus.

Curcumin enhances the activity of fluconazole against Cryptococcus gattii-induced cryptococcosis infection in mice.

AIM: The aim of this study was to investigate the in vitro and in vivo activities of pure curcumin, as well as its combination with fluconazole, against Cryptococcus gattii. METHODS AND RESULTS: The minimal inhibitory concentrations (MIC) of curcumin and its interactions with fluconazole against C. gattii were assessed in vitro using standard methods. This same combination was used to treat C. gattii-induced cryptococcosis in mice. The behavioural and functional assessment of the mice during treatment was also performed. The average MIC for curcumin was 19·8 µg ml(-1) . Its combination with fluconazole resulted in FICΣ (fractional inhibitory concentration index) values between 0·79 and 2·29. Curcumin (alone or combined with fluconazole) significantly reduced pulmonary damage and fungal burden in the brain. No colonies were found in the brain following combination treatment, which was also confirmed by the improved behaviour of mice. CONCLUSIONS: The combination therapy with curcumin and fluconazole was the most effective among the treatments tested, as in addition to reducing the fungal burden and damage on lung tissues, it was able to eliminate the fungal burden in the brain, enhancing the survival of mice. SIGNIFICANCE AND IMPACT OF THE STUDY: This study points to the possibility of using curcumin in combination with fluconazole as a clinical treatment of cryptococcosis.

Lactate and heart rate variability threshold during resistance exercise in the young and elderly.

This purpose of this study was to: 1) determine the intensity corresponding to anaerobic threshold (AT) during a discontinuous resistance exercise protocol in healthy young and elderly subjects by analyzing heart rate variability (HRV) and blood lactate (BL) and 2) investigate the effect of aging on these variables. A total of 28 individuals, 14 young and 14 elderly healthy men underwent one-repetition maximum (1RM) testing to determine maximum load on the leg press. Discontinuous resistance exercise testing was initiated at 10% of the 1RM with subsequent increases of 10%. The load corresponding to AT was approximately 30% 1RM in both groups. The determination of AT by HRV was associated with BL responses (p<0.01). While HRV indexes decreased with increasing of loads in both groups, the elderly had lower values at loads below AT (p<0.05). Additionally, BL increased sharply after the load corresponding to AT in both groups, although elderly subjects showed the lowest values (p<0.05). In conclusion, HRV is an effective tool for determining AT, which was approximately 30% 1RM under the testing procedures included in the present study. Furthermore, there was a marked change in autonomic function, with gradual vagal withdrawal followed by sympathetic activation. These responses were lower in elderly subjects.

Influence of different media, incubation times, and temperatures for determining the MICs of seven antifungal agents against Paracoccidioides brasiliensis by microdilution.

MIC assays with Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, had been conducted with variable protocols, employing both macrodilution and microdilution tests and including differences in inoculum preparation, media used, incubation periods, and temperatures. Twenty-one clinical and environmental isolates of Paracoccidioides were tested using amphotericin B, itraconazole, ketoconazole, fluconazole, sulfamethoxazole, sulfamethoxazole-trimethoprim, and terbinafine, according to the National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, document M27-A2, 2002), with modifications such as three medium formulations (RPMI 1640 medium, McVeigh and Morton [MVM] medium, and modified Mueller-Hinton [MMH] medium), two incubation temperatures (room temperature [25 to 28 °C] and 37 °C), and three incubation periods (7, 10, and 15 days). The antifungal activities were also classified as fungicidal or fungistatic. The best results were obtained after 15 days of incubation, which was chosen as the standard incubation time. The MICs for most individual isolates grown for the same length of time at the same temperature varied with the different media used (P < 0.05). Of the isolates, 81% showed transition from the yeast to the mycelial form in RPMI 1640 medium at 37 °C, independent of the presence of antifungals. MMH medium appears to be a suitable medium for susceptibility testing of antifungal drugs with P. brasiliensis, except for sulfamethoxazole and the combination of sulfamethoxazole-trimethoprim, for which the MVM medium yielded better results. The incubation temperature influenced the MICs, with, in general, higher MICs at 25 °C (mycelial form) than at 37 °C (P < 0.05). Based on our results, we tentatively propose a microdilution assay protocol for susceptibility testing of antifungal drugs against Paracoccidioides.

Outbreak of Neisseria meningitidis C in workers at a large food-processing plant in Brazil: challenges of controlling disease spread to the larger community.

SUMMARYAn outbreak of meningococcal disease (MD) with severe morbidity and mortality was investigated in midwestern Brazil in order to identify control measures. A MD case was defined as isolation of Neisseria meningitidis, or detection of polysaccharide antigen in a sterile site, or presence of clinical purpura fulminans, or an epidemiological link with a laboratory-confirmed case-patient, between June and August 2008. In 8 out of 16 MD cases studied, serogroup C ST103 complex was identified. Five (31%) cases had neurological findings and five (31%) died. The attack rate was 12 cases/100 000 town residents and 60 cases/100 000 employees in a large local food-processing plant. We conducted a matched case-control study of eight primary laboratory-confirmed cases (1:4). Factors associated with illness in single variable analysis were work at the processing plant [matched odds ratio (mOR) 22, 95% confidence interval (CI) 2·3-207·7, P<0·01], and residing <1 year in Rio Verde (mOR 7, 95% CI 1·11-43·9, P<0·02). Mass vaccination (>10 000 plant employees) stopped propagation in the plant, but not in the larger community.

Outbreak of acute gastroenteritis in young children with death due to rotavirus genotype G9 in Rio Branco, Brazilian Amazon region, 2005.

BACKGROUND: An epidemic of acute gastroenteritis occurred in Rio Branco City, Acre State, in Brazil's Amazon region in 2005. An investigation was conducted to confirm the etiology and identify possible risk factors for death. METHODS: Rio Branco municipality surveillance data for the period May to October 2005 were reviewed. In a case-control study, children who died following acute gastroenteritis were compared to age-matched controls with acute gastroenteritis who survived. Rotavirus A (RV-A) was investigated in 799 stool samples and genotyped by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The cumulative incidence of diarrhea in children aged <5 years was 21%. A fatal outcome was significantly associated with uncovered household water storage containers. RV-A was identified in 88% of samples and G9 was the prevalent genotype (71%). CONCLUSIONS: Oral rehydration solution and boiling or chlorinating drinking water likely limited mortality. This epidemic was caused by RV-A genotype G9. After the outbreak, a rotavirus vaccine was introduced into the official childhood immunization schedule in Brazil.

Correlation of the in vitro antifungal drug susceptibility with the in vivo activity of fluconazole in a murine model of cerebral infection caused by Cryptococcus gattii.

Forty Cryptococcus gattii strains were submitted to antifungal susceptibility testing with fluconazole, itraconazole, amphotericin B and terbinafine. The minimum inhibitory concentration (MIC) ranges were 0.5-64.0 for fluconazole, <0.015-0.25 for itraconazole, 0.015-0.5 for amphotericin B and 0.062-2.0 for terbinafine. A bioassay for the quantitation of fluconazole in murine brain tissue was developed. Swiss mice received daily injections of the antifungal, and their brains were withdrawn at different times over the 14-day study period. The drug concentrations varied from 12.98 to 44.60 µg/mL. This assay was used to evaluate the therapy with fluconazole in a model of infection caused by C. gattii. Swiss mice were infected intracranially and treated with fluconazole for 7, 10 or 14 days. The treatment reduced the fungal burden, but an increase in fungal growth was observed on day 14. The MIC for fluconazole against sequential isolates was 16 µg/mL, except for the isolates obtained from animals treated for 14 days (MIC = 64 µg/mL). The quantitation of cytokines revealed a predominance of IFN-γ and IL-12 in the non-treated group and elevation of IL-4 and IL-10 in the treated group. Our data revealed the possibility of acquired resistance during the antifungal drug therapy.

In vitro activities of four antifungal drugs against Trichophyton rubrum isolates exhibiting resistance to fluconazole.

Thirty-two clinical isolates of Trichophyton rubrum exhibiting resistance to fluconazole [minimum inhibitory concentrations (MICs) > or = 64 microg ml(-1)] were selected to test the antifungal activity of ketoconazole, itraconazole, griseofulvin and terbinafine. We followed the guidelines of the National Committee for Clinical Laboratory Standards for testing filamentous fungi. The strains Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC 40051) and Trichophyton mentagrophytes (ATCC 40004) were included for quality control. The microdilution plates were incubated at 28 degrees C and were read visually after 7 days of incubation and endpoint determination readings were performed visually. The MIC ranges for the four antifungals were: 0.0625-2 microg ml(-1) for ketoconazole, 0.25-2.0 microg ml(-1) for griseofulvin, < or =0.031-1.0 microg ml(-1) for itraconazole and < or =0.031 microg ml(-1) for terbinafine (for all tested isolates). Terbinafine was the most potent drug against T. rubrum, in vitro, followed by itraconazole, ketoconazole and griseofulvin. Much work is still needed to correlate the MICs of these drugs with clinical outcomes to develop interpretative breakpoints for T. rubrum and other dermatophytes.

In vitro cytotoxicity activity on several cancer cell lines of acridone alkaloids and N-phenylethyl-benzamide derivatives from Swinglea glutinosa (Bl.) Merr.

The methanol extract from the stems and fruits of Swinglea glutinosa (Rutaceae) afforded 11 known acridone alkaloids and three N-phenylethyl-benzamide derivatives, glycocitrine-IV, 1,3,5-trihydroxy-4-methoxy-10-methyl-2,8-bis(3-methylbut-2-enyl)acridin-9(10H)-one, 1,3,5- trihydroxy-2,8-bis(3-methylbut-2-enyl)-10-methyl-9-acridone, citbrasine, citrusinine-II, citrusinine-I, 5-dihydroxyacronycine, pyranofoline, 3,4-dihydro-3,5,8-trihydroxy-6-methoxy-2,2,7-trimethyl-2H-pyrano[2,3-a]acridin-12(7H)-one, 2,3-dihydro-4,9-dihydroxy-2-(2-hydroxy-propan-2-yl)-11-methoxy-10-methylfuro[3,2-b]acridin-5(10H)-one, bis-5-hydroxyacronycine, N-(2-{4-[(3,7-dimethylocta-2,6-dien-1-yl)oxy]phenyl}ethyl)benzamide, N-(2-{4-[(3,7-dimethyl-4-acethyl-octa-2,6-dien-1-yl)oxy]phenyl}ethyl)benzamide, and severine acetate. All compounds isolated were examined for their activity against three cancer cell lines: human lung carcinoma (COR-L23), human breast adenocarcinoma (MCF7), human melanoma (C32), and normal human fetal lung cell line, MRC-5. The acridones tested exhibited weak cytotoxicity but the amides showed moderate nonselective cytotoxic activity.

In vitro antifungal oral drug and drug-combination activity against onychomycosis causative dermatophytes.

We present the results of studies of the in vitro susceptibility of 52 isolates of Trichophyton rubrum and 40 of Trichophyton mentagrophytes to griseofulvin, terbinafine, itraconazole, ketoconazole, fluconazole and cyclopiroxolamine. All test strains were recovered from patients with toe nail onychomycosis and the minimum inhibitory concentration (MIC) of each antifungal against both species was individually assessed. In addition, we investigated the MIC of the combination of cyclopiroxolamine and itraconazole and cyclopiroxolamine and ketoconazole. The NCCLS approved procedure M38-A as modified by Santos and Hamdan was employed. The studies of the two drug combinations were conducted with a checkerboard design. Analysis of the data revealed that terbinafine was the most effective in vitro against all isolates, followed in order by itraconazole, cyclopiroxolamine, ketoconazole and fluconazole. We observed no significant difference in the in vitro susceptibility profiles between either species to any of the antifungals (P<0.05). Our in vitro results confirm that terbinafine is the most effective of the antifungals included in this study. Furthermore, synergistic interactions were found in the two drug combinations with all of the dermatophyte test isolates. The latter results are in agreement with clinical data that show synergism between oral and topical antifungals in the treatment of onychomycosis.

Establishing a method of inoculum preparation for susceptibility testing of Trichophyton rubrum and Trichophyton mentagrophytes.

A total of 92 clinical isolates of dermatophytes (52 of Trichophyton rubrum and 40 of Trichophyton mentagrophytes) were selected for testing with six antifungal drugs (terbinafine, griseofulvin, clotrimazole, miconazole, isoconazole, and fluconazole) and two pairs of drug combinations (ketoconazole-cyclopiroxolamine and itraconazole-cyclopiroxolamine). Two methods of inoculum preparation for susceptibility testing were evaluated that used (i) inocula consisting only of microconidia of dermatophytes filtered in Whatman filter model 40 and (ii) unfiltered inocula consisting of hyphae and microconidia. We followed the recommendations of approved document M38-A of CLSI (formerly NCCLS) with some adaptations, including an incubation period of 7 days and an incubation temperature of 28 degrees C. Reference strains of Candida parapsilosis, Candida krusei, Trichophyton rubrum, and Trichophyton mentagrophytes were included as quality-control strains. MICs were consistently higher (usually 1 to 2 dilutions for drugs tested individually) when nonfiltered inocula were tested (P < 0.01) except for terbinafine. Larger MICs were seen when testing drugs with nonfiltered inocula. The curves of drug interaction were used to analyze the reproducibility of the test, and it was shown that high levels of reproducibility were achieved using the methodology that included the filtration step. The standardization of methodologies is the first step to yield reliability of susceptibility testing and to proceed with clinical laboratory studies to correlate MICs with clinical outcomes.

Contagem de Staphylococcus sp. e deteccão de enterotoxinas estafilocócicas e toxina da síndrome do choque tóxico em amostras de leite cru refrigerado / Staphylococcus sp. counting and detection of staphylococcal enterotoxins and toxic shock toxin syndrome from cooled raw milk

Analisaram-se 80 amostras de leite cru refrigerado a 4ºC e estocado por 48 horas em tanques refrigeradores de propriedades rurais do estado de Minas Gerais quanto à contagem e identificacão de Staphylococcus sp. e deteccão de enterotoxinas estafilocócicas (SE) e da toxina da síndrome do choque tóxico (TSST-1). Staphylococcus sp. foi detectado em 100 por cento das amostras de leite de tanque refrigerador em contagens que variaram de 1,0 10(5) a 2,5 10(7) UFC/ml (média = 5,60 log UFC/ml; s = 0,53 e CV = 9,5 por cento). Isolaram-se e identificaram-se 436 estirpes como: S. aureus, S hyicus, S. epidermidis, S. intermedius, S. cohnii, S. sciuri, S. schleirferi e S. delphini. As estirpes de mesmo perfil bioquímico, oriundas da mesma amostra, foram agrupadas (pools) e induzidas a produzir SE e TSST-1. A deteccão dessas enterotoxinas foi feita pelo método optimum sensitivity plate, usando-se técnica de celofane sobre ágar. Identificou-se a producão de SEA, SEB, SEC, SED e de TSST-1 em percentuais variados. Dos 138 pools preparados, 91 produziram, pelo menos, uma toxina isoladamente ou em associacão a outras toxinas. Dos pools enterotoxigênicos, 24,6 por cento eram coagulase positiva e 41,3 por cento, coagulase negativa. A confirmacão de estirpes enterotoxigênicas de Staphylococcus coagulase negativa isoladas de amostras de leite é importante em relacão à saúde pública.

Contagem de Staphylococcus sp. e deteccão de enterotoxinas estafilocócicas e toxina da síndrome do choque tóxico em amostras de leite cru refrigerado / Staphylococcus sp. counting and detection of staphylococcal enterotoxins and toxic shock toxin syndrome from cooled raw milk

Analisaram-se 80 amostras de leite cru refrigerado a 4°C e estocado por 48 horas em tanques refrigeradores de propriedades rurais do estado de Minas Gerais quanto à contagem e identificação de Staphylococcus sp. e detecção de enterotoxinas estafilocócicas (SE) e da toxina da síndrome do choque tóxico (TSST-1). Staphylococcus sp. foi detectado em 100% das amostras de leite de tanque refrigerador em contagens que variaram de 1,0 × 105 a 2,5 × 107 UFC/ml (média = 5,60 log UFC/ml; s = 0,53 e CV = 9,5%). Isolaram-se e identificaram-se 436 estirpes como: S. aureus, S hyicus, S. epidermidis, S. intermedius, S. cohnii, S. sciuri, S. schleirferi e S. delphini. As estirpes de mesmo perfil bioquímico, oriundas da mesma amostra, foram agrupadas (pools) e induzidas a produzir SE e TSST-1. A detecção dessas enterotoxinas foi feita pelo método optimum sensitivity plate, usando-se técnica de celofane sobre ágar. Identificou-se a produção de SEA, SEB, SEC, SED e de TSST-1 em percentuais variados. Dos 138 pools preparados, 91 produziram, pelo menos, uma toxina isoladamente ou em associação a outras toxinas. Dos pools enterotoxigênicos, 24,6% eram coagulase positiva e 41,3%, coagulase negativa. A confirmação de estirpes enterotoxigênicas de Staphylococcus coagulase negativa isoladas de amostras de leite é importante em relação à saúde pública.(AU)

In order to count and identify Staphylococcus sp., the detection of the Staphylococcal enterotoxins (SE) and toxic shock toxin syndrome (TSST-1), 80 raw milk samples cooled at 4°C and stored in bulk tanks for 48 hours in different farms from Minas Gerais State were analyzed. Staphylococcus sp. was observed in all samples and the counts varied from 1.0 × 105 to 2.5 × 107 CFU/ml (mean = 5.60 log CFU/ml; sd = 0.53 and CV = 9.46 %). A total of 436 strains of Staphylococcus were isolated and identified as S. aureus, S hyicus, S. epidermidis, S. intermedius, S. cohnii, S. sciuri, S. schleirferi and S. delphini. Strains showing identical biochemical profile, from the same sample, were grouped into a pool and them were induced to produce SE and TSST-1. The detection of toxins was made by the OSP (Optimum Sensivity Plate) method and the cellophane-over-agar technique. It was identified SEA, SEB, SEC, SED and TSST-1 in different percentages. From the 138 formed pools, 91 produced, at least, one or more toxin, including TSST-1. From the enterotoxigenic pools, 24.6% were coagulase positive, while 41.3% were negative. The presence of entorotoxigenic negative coagulase Staphylococcus strains isolated from milk samples is important in relation to public health.(AU)

Evaluation of broth microdilution antifungal susceptibility testing conditions for Trichophyton rubrum.

Fifty clinical isolates of Trichophyton rubrum were selected to test with ketoconazole, fluconazole, itraconazole, griseofulvin, and terbinafine by following the National Committee for Clinical Laboratory Standards susceptibility testing guidelines for filamentous fungi (M38-A). In addition, other susceptibility testing conditions were evaluated: (i) three medium formulations including RPMI 1640 (standard medium), McVeigh & Morton (MVM), and Sabouraud dextrose broth (SDB); (ii) two incubation temperatures (28 and 35 degrees C); and (iii) three incubation periods (4, 7, and 10 days). The strains Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC 40051), and Trichophyton mentagrophytes (ATCC 40004) were included as quality controls. All isolates produced clearly detectable growth only after 7 days of incubation. MICs were significantly independent of the incubation temperature (28 or 35 degrees C) (P < 0.05). Different incubation periods resulted in MICs which were consistently different for each medium when azoles and griseofulvin were tested (P < 0.05). MICs obtained from different media at the same incubation time for the same isolate were significantly different when azoles and griseofulvin were tested (P < 0.05). MICs were consistently higher (usually 1 to 2 dilutions) with RPMI than with MVM or SDB (P < 0.05). When terbinafine was tested, no parameter had any influence on MICs (P < 0.05). RPMI standard medium appears to be a suitable testing medium for determining the MICs for T. rubrum. MICs obtained at different incubation times need to be correlated with clinical outcome to demonstrate which time has better reliability.