A comparison of methods for the detection of Candida antigens. Evaluation of a new latex reagent.

The sensitivity of various serological techniques for the detection of C. albicans cytoplasmic antigen (Ag) in buffer and serum diluents was compared, with special reference to variations of the ELISA method and a new microtitre latex particle agglutination (MLA) test. Of the assays evaluated, immunodiffusion, counterimmunoelectrophoresis and slide latex particle agglutination (SLA) were the least sensitive. ELISA tests were more sensitive (50-150 ng/ml Ag in buffer) although sensitivity decreased in serum or heat-inactivated serum (HIS) (250 ng/ml-4 micrograms/ml). The new MLA test had better sensitivity (16 ng/ml Ag in buffer) than any of the ELISA tests and was unaffected by the presence of serum or HIS (2.5 and 20 ng/ml Ag respectively). MLA seems worthy of further evaluation as an alternative to ELISA for use in antigen detection systems in general and for the serodiagnosis of systemic candidosis in particular.

Detection of Candida precipitins. a comparison of double diffusion and counter immunoelectrophoresis.

The optimum conditions for detection of Candida precipitins by double diffusion and counter immunoelectrophoresis have been established. Counter immunoelectrophoresis was shown to be more sensitive than double diffusion, and its application to routine tests for Candida precipitins to cytoplasmic antigen is recommended.

The incidence of pathogenic yeasts among open-heart surgery patients-the value of prophylaxis.

The normal levels of commensal yeasts in patients undergoing open-heart surgery are established and the effect of antifungal prophylaxis is assessed. Mouth swabs and feces were taken for culture from patients on admission to hospital and 1,2, and 3 weeks postoperatively. Eighty-seven patients who received normal treatment and 50 patients who were given oral and topical antifungal prophylaxis commencing 12 days before hospitalization were studied. Yeast pathogens, mainly Candida albicans, were isolated from 42 (48.3 per cent) of the normal group on admission. There was a marked increase in the incidence and quantities of yeasts isolated from patients in the immediate postoperative period. The incidence and levels of yeasts in patients receiving antifungal prophylaxis was considerably reduced both on admission and postoperatively. The risk of Candida sepsis in open-heart surgery patients with high levels of commensal yeasts is discussed and the possibility of routine antifungal prophylaxis raised.

Distribution of pathogenic yeasts and humoral antibodies to candida among hospital inpatients.

The frequencies of the carriage of yeast pathogens and of serum precipitins to a variety of candida antigens among 254 patients generally tended to increase with the length of the patient's stay in hospital. This trend was observed even though none of the patients investigated showed signs or symptoms of superficial or systemic candidosis. The extent of the general trend varied considerably between subgroups of patients within the general categories of 'surgical' and 'nonsurgical' inpatients. Increases in both frequencies and quantities of yeasts in the mouth were most evident postoperatively among patients who underwent open-heart surgery and among nonsurgical patients who received antibiotics or steroids in hospital. The frequency of precipitins to Candida albicans cytoplasmic antigens in the absence of candidosis rose overall from 11% of 217 sera obtained within 24 hours of admission to 35% of 85 sera obtained five to 11 days after admission or operation. These 'false positive' antibodies were thought to arise after transient yeast overgrowth in the gut at the time of an acute illness or immediately after surgery. The study adds further data to documented examples of 'false positive' candida antibodies and indicates the need for care in the diagnostic interpretation of candida precipitin test results among groups of patients at risk of yeast overgrowth during their hospital stay.

Rapid diagnosis of vaginal candidosis by latex particle agglutination.

Vaginal swabs from women who on clinical evidence were thought to have vaginal candidosis were examined for yeasts by conventional laboratory methods (microscopy and culture) and also assayed for Candida antigens using a rapid (3 min) slide latex particle agglutination tests. Results showed that a diagnosis of vaginal candidosis based on clinical criteria alone is unreliable: only half of the women were subsequently confirmed as having candidosis by microscopy and culture. The new slide latex particle agglutination test gave better results, with 100% specificity, 80% sensitivity, high predictive values (greater than or equal to 91%), and an overall diagnostic efficiency of 93%. From the results of this preliminary study, slide latex particle agglutination looks a promising, rapid alternative to conventional laboratory methods for confirming a clinical diagnosis of vaginal candidosis and has the considerable advantage that it can be conveniently used in a clinical setting.

Prevalence of pathogenic yeasts and humoral antibodies to candida in diabetic patients.

The prevalence of oral yeasts and humoral precipitating antibodies to candida was estimated in 204 unselected diabetic patients (172 outpatients and 32 inpatients). Yeasts, mainly Candida albicans, were isolated from the mouths of 41% of the outpatients and precipitins were found in 17.5% although none of the patients had clinically overt candidiasis. The extent of oral yeast colonisation and incidence of antibodies was not related to their antidiabetic treatment or to the duration of their diabetes. It was, however, related to the blood glucose and urine sugar levels at the time they were sampled, the highest incidence being among the diabetic inpatients with high blood glucose levels at the time of sampling and the lowest among outpatients with normal blood glucose levels at the time of sampling. There was no such correlation when diabetic control over the previous 12-month period was considered.

Detection of Aspergillus fumigatus PCR products by a microtitre plate based DNA hybridisation assay.

AIMS: To develop a DNA based plate hybridisation assay for the detection of polymerase chain reaction (PCR) products amplified from Aspergillus fumigatus DNA; and to determine the sensitivity of this technique and compare it with Southern blotting. METHODS: A half-log dilution series of DNA extracted from A fumigatus was amplified with specific primers, one of which was 5' end labelled with biotin. PCR products were subsequently detected by agarose gel electrophoresis, Southern blotting, and binding of the products to a streptavidin coated microtitre well, followed by non-radioactive colorimetric detection. Amplification was carried out 10 times for each DNA dilution and a plot of initial DNA concentration against signal intensity was made. RESULTS: A DNA concentration of 1.5 pg could be detected by agarose gel electrophoresis and Southern blotting with a non-radioactively labelled aspergillus specific probe; 1.5 pg was detectable by streptavidin binding of the PCR products to a microtitre plate. The signal from the microtitre plate detection was proportional to the amount of DNA in the PCR reaction on a log-log scale between 100 and 1 pg of DNA. CONCLUSIONS: A DNA based plate hybridisation assay for the detection of A fumigatus PCR products is as sensitive as Southern blotting. However, results are obtained in three hours rather than the three days required for agarose gel electrophoresis, blotting, hybridisation, and detection.

Use of the Pastorex aspergillus antigen latex agglutination test for the diagnosis of invasive aspergillosis.

AIMS: To evaluate the Pastorex aspergillus antigen latex agglutination test for the diagnosis of invasive aspergillosis in patients undergoing liver or bone marrow transplantation. METHODS: Serum samples were taken at least twice weekly post-transplant and tested for Aspergillus antigen. Latex agglutination test results were compared with microbiological examination of respiratory, urine and bile specimens. Serum samples from liver transplant patients were also tested for antibodies to Aspergillus fumigatus by counter immunoelectrophoresis. RESULTS: Eight of the 91 patients studied developed invasive aspergillosis. Positive latex agglutination tests were obtained in eight of 187 (4.3%) serum samples from four of these eight patients. The other four patients with invasive aspergillosis gave consistently negative latex agglutination tests. A positive latex agglutination test was the first indication of invasive aspergillosis in two patients; these patients were already on amphotericin B. Positive latex agglutination tests were the only evidence of invasive aspergillosis in one patient who subsequently died of the infection. False positive latex agglutination tests were obtained in five of 83 (6%) patients with no evidence of invasive aspergillosis and misleading positive cultures seen in nine of 83 (10.8%). No antibodies were detected in three of four liver transplant patients with invasive aspergillosis. Conversely, antibodies were detected in 63 of 262 (24%) serum samples from 43 liver transplant patients with no evidence of invasive aspergillosis; one of these patients had an antibody titre of 1:2 on four separate occasions. CONCLUSIONS: The Pastorex aspergillus antigen latex agglutination test, when used alone, lacks sensitivity and specificity for the early diagnosis of invasive aspergillosis. A diagnosis was made in all patients with invasive aspergillosis when both culture and antigen tests were performed although using these criteria a false positive diagnosis would have been made in 13 of 83 (15.6%) patients. Microbiological and serial serological investigations for antigen should both be performed and the results considered in conjunction with radiological and clinical evidence.

A two year global evaluation of the susceptibility of Candida species to fluconazole by disk diffusion.

The in-vitro activity of fluconazole against 46,831 yeast isolates collected over a two-year period from 57 laboratories in 33 countries worldwide was assessed using a disc diffusion method. Candida albicans was the organism isolated most frequently, accounting for 68.6% of the total number of isolates. C. glabrata, C. tropicalis, C parapsilosis and C. krusei and Cryptococcus neoformans represented 9.9, 4.7, 4.3, 1.9, and 1.4% of isolates respectively during the 2 year period and rates varied markedly between countries. In 1999 data blood isolates represented 4.9% of all isolates and intensive care unit isolates represented 9.9%. In both the 1998 and 1999 data, 99% of C. albicans were fully susceptible (S) to fluconazole, and 95.6% of all species of yeasts tested were S or susceptible-dose dependent (S-DD) to fluconazole. No emerging trends of resistance were noted with any of the Candida spp. tested as 96% of all isolates retained susceptibility (S or S-DD) to this agent.

Antibodies to Candida after operations on the heart.

The occurrence of yeasts and antibodies to yeasts was studied in patients undergoing open-heart surgery without antifungal prophylaxis, and in a similar group receiving antifungal prophylaxis. An association was demonstrated between the occurrence of commensal yeasts and the appearance of antibodies. None of the patients developed overt systemic or superficial yeast infection. The antigenic stimulus for the post-operative production of antibodies appeared to be the increase in the yeast flora that occurred shortly after operation. When the commensal yeast population was suppressed by antifungal antibiotics, the antibody response was also reduced. The implications of these findings in the interpretation of serological tests for diagnosis of systemic yeast infections are discussed.

Current and future approaches to antimycotic treatment in the era of resistant fungi and immunocompromised hosts.

Due to the ever-increasing number of immunocompromised patients, both localised and life-threatening systemic fungal infections are on the increase. Conventional treatment is of limited help, not in the least due to a less optimum benefit-to-risk ratio. Moreover, emerging pathogens with reduced antimicrobial susceptibility and the development of resistance in Candida albicans form a new challenge. Fortunately, conventional antimycotics have been improved and entirely new ones are on the horizon as well as alternative approaches such as immunoreconstitution.

Scedosporium prolificans, a multi-resistant fungus, from a U.K. AIDS patient.

We report the first isolation of Scedosporium prolificans (inflatum) in the U.K. The patient, with advanced AIDS and neutropenia, had respiratory tract colonisation over many months without invasive disease despite neutropenia, while on itraconazole therapy. The organism was resistant in vitro to all licensed systemic antifungal agents tested.

The oncogenic potential of Candida in the female genital tract.

The possible role of Candida species in carcinogenesis at the uterine cervix was investigated in 226 females attending a colposcopy clinic. Approximately 34% of the 226 subjects harbored Candida species in cervical/vaginal secretions, but there was no association with any particular histologic abnormality. Two independent analytical procedures were used for strain discrimination of the isolates of C. albicans, but again no relationship was found between individual strains and histologic diagnoses. Only three C. glabrata strains were isolated, but they were all in association with cervical intraepithelial neoplasia (CIN) II or III. A total of 18 strains of C. albicans, one C. glabrata and one C. parapsilosis all inhibited the formation of the nitrosamine nitrosodimethylamine (NDMA) from precursors. Furthermore, C. albicans strains did not convert NDMA to carcinogenic metabolites. The results of this study do not suggest that C. albicans has a role in cervical carcinogenesis.

Testing of antifungal combinations against yeasts and dermatophytes.

BACKGROUND: Fungal infections of the nail are a common and chronic problem. The main pathogens responsible for onychomycosis are dermatophytes, yeasts and moulds. Despite significant improvements, approximately 20% of patients with onychomycosis still fail on antifungal therapy. The successful exploitation of drug synergy may provide a useful approach to improve cure rates. METHODS: The minimum inhibitory concentrations (MIC(80)) were recorded for pathogens that are most frequently responsible for onychomycosis against combinations of several antifungal agents, namely, fluconazole, itraconazole, terbinafine and amorolfine. Fractional inhibitory concentrations (FICs) were then calculated from the MIC(80) results and the FIC values for each drug in the combinations added to determine the degree of synergy. A combined value of <1 was taken to suggest synergy; a value of 1-2 indicated an additive effect or indifference; and a combined FIC value of >2 was taken to suggest antagonism. RESULTS: Overall, 46% of amorolfine combinations showed results suggestive of synergy, with the most synergistic results seen against dermatophytes (54%) and moulds (52%). CONCLUSIONS: Some combinations of drugs may have synergistic activity in vitro; however, the importance of this in a clinical setting is yet to be established, and more studies are justified.

Criteria for the diagnosis of vaginal candidosis: evaluation of a new latex agglutination test.

The value of current diagnostic methods (clinical examination, microscopy, mycological culture) was compared with a new slide latex agglutination (SLA) test for the diagnosis of vaginal candidosis in 224 women attending a genito-urinary medicine clinic. Candida albicans was isolated from 44 (19.6%) of women but just less than half of this number (21) had clinical candidosis. Clinical examination correctly identified 81% of women with candidosis but the predictive value of a positive clinical diagnosis was only 51.5%. Microscopical examination of a Gram-stained vaginal smear was also inaccurate and yeasts were seen in smears from only 42.9% of women with candidosis. Cultures were positive in 95.2% of those with candidosis, but 54.6% of women with positive cultures showed only commensal carriage. Overall, the SLA test was more successful. It was as sensitive (81%) as clinical diagnosis in identifying patients with candidosis but it was more specific (98.5%) than the other diagnostic criteria and had the highest overall test efficiency (96.8%).

Double blind, randomised study of continuous terbinafine compared with intermittent itraconazole in treatment of toenail onychomycosis. The LION Study Group.

OBJECTIVE: To compare the efficacy and safety of continuous terbinafine with intermittent itraconazole in the treatment of toenail onychomycosis. DESIGN: Prospective, randomised, double blind, double dummy, multicentre, parallel group study lasting 72 weeks. SETTING: 35 centres in six European countries. SUBJECTS: 496 patients aged 18 to 75 years with a clinical and mycological diagnosis of dermatophyte onychomycosis of the toenail. INTERVENTIONS: Study patients were randomly divided into four parallel groups to receive either terbinafine 250 mg a day for 12 or 16 weeks (groups T12 and T16) or itraconazole 400 mg a day for 1 week in every 4 weeks for 12 or 16 weeks (groups I3 and I4). MAIN OUTCOME MEASURES: Assessment of primary efficacy at week 72 was mycological cure, defined as negative results on microscopy and culture of samples from the target toenail. RESULTS: At week 72 the mycological cure rates were 75.7% (81/107) in the T12 group and 80. 8% (80/99) in the T16 group compared with 38.3% (41/107) in the I3 group and 49.1 % (53/108) in the I4 group. All comparisons (T12 v I3, T12 v I4, T16 v I3, T16 v I4) showed significantly higher cure rates in the terbinafine groups (all P<0.0001). Also, all secondary clinical outcome measures were significantly in favour of terbinafine at week 72. There were no differences in the number or type of adverse events recorded in the terbinafine or itraconazole groups. CONCLUSION: Continuous terbinafine is significantly more effective than intermittent itraconazole in the treatment of patients with toenail onychomycosis.

Short term treatment of dermatophyte onychomycosis with terbinafine.

OBJECTIVE: To evaluate the effect of short term treatment with terbinafine on dermatophytosis. DESIGN: Multicentre, randomised, double blind placebo controlled trial of 250 mg/day terbinafine for 12 weeks in dermatophyte onychomycosis. SETTING: Eight dermatology centres in the United Kingdom. PATIENTS: 112 patients (mean age 44, range 19-78), 99 with mycologically proved toenail infections and 13 with fingernail infections, of whom eight were subsequently excluded and 19 failed to complete the study. INTERVENTION: Terbinafine 250 mg daily or placebo for 12 weeks. Follow up for 36 weeks after stopping treatment. MAIN OUTCOME MEASURES: Mycological cure (negative results on microscopy and culture) and clinical cure at the end of follow up, adverse events, and biochemical and haematological variables at monthly intervals during treatment. RESULTS: After follow up 82% (37/45) (95% confidence interval 68% to 92%) mycological cure and 69% clinical cure were recorded for evaluable patients treated with terbinafine for toenail infection and 71% (5/7) (30% to 96%) mycological cure and clinical cure for those treated for fingernail infection. The corresponding values for those treated with placebo were 12% (3% to 31%) mycological cure and no clinical cure for toenail infections and 33% (1% to 91%) mycological cure and no clinical cure for fingernail infections. On an intention to treat basis for toenail infections the figures were 73% (38/52) (58% to 85%) mycological cure for terbinafine compared with 6% (0% to 30%) for placebo (p less than 0.007). Two withdrawals were related to adverse events with terbinafine, and there were no significant abnormal laboratory test results. CONCLUSION: 12 weeks' terbinafine is effective and safe treatment for nail dermatophytosis.

Comparison of terbinafine and clotrimazole in treating tinea pedis.

OBJECTIVE: To compare the efficacy and safety of terbinafine 1% cream and clotrimazole 1% cream in the treatment of tinea pedis. DESIGN: Multicentre, double blind parallel group study. SETTING: 32 general practices and one hospital. PATIENTS: 256 patients with mycologically confirmed tinea pedis. Of the 211 patients evaluable, 107 were randomised to terbinafine (75 male, 32 female; mean (range) age 40 (12-81) years) and 104 to clotrimazole (79 male, 25 female; mean (range) age 36 (12-71) years). INTERVENTIONS: Terbinafine 1% cream applied twice daily for one week and inert cream applied twice daily for the next three weeks. Clotrimazole 1% cream applied twice daily for four weeks. MAIN OUTCOME MEASURES: Mycological cure (negative results on microscopy and culture) and effective treatment (mycological cure plus no or minimal signs and symptoms) measured at weeks 1, 2, 3, 4, and 6. RESULTS: At week four rates of mycological cure were 93.5% for terbinafine and 73.1% for clotrimazole (p = 0.0001); and at week six 97.2% for terbinafine and 83.7% for clotrimazole (p = 0.001). Rates of effective treatment at week 4 were 89.7% for terbinafine and 58.7% for clotrimazole (p = 0.0001); and 89.7% for terbinafine and 73.1% for clotrimazole (p = 0.002) at week 6. CONCLUSION: These results indicate that a one week course of terbinafine 1% cream is more effective in the treatment of tinea pedis than a four week course of clotrimazole 1% cream, both in terms of mycological cure and effective treatment.