Implications of the EUCAST Trailing Phenomenon in Candida tropicalis for the In Vivo Susceptibility in Invertebrate and Murine Models.

Candida tropicalis isolates often display reduced but persistent growth (trailing) over a broad fluconazole concentration range during EUCAST susceptibility testing. Whereas weak trailing (<25% of the positive growth control) is common and found not to impair fluconazole efficacy, we investigated if more pronounced trailing impacted treatment efficacy. Fluconazole efficacy against two weakly (≤25% growth), two moderately (26% to 50% growth), and one heavily (>70% growth) trailing resistant isolate and one resistant (100% growth) isolate were investigated in vitro and in vivo (in a Galleria mellonella survival model and two nonlethal murine models). CDR1 expression levels and ERG11 sequences were characterized. The survival in fluconazole-treated G. mellonella was inversely correlated with the degree of trailing (71% to 9% survival in treatment groups). In mice, resistant and heavily trailing isolates responded poorly to fluconazole treatment. CDR1 expression was significantly higher in trailing and resistant isolates than in wild-type isolates (1.4-fold to 10-fold higher). All isolates exhibited ERG11 wild-type alleles. Heavily trailing isolates were less responsive to fluconazole in all in vivo models, indicating an impact on fluconazole efficacy. CDR1 upregulation may have contributed to the observed differences. Moderately trailing isolates responded less well to fluconazole in larvae only. This confirms clinical data suggesting fluconazole is effective against infections with such isolates in less severely ill patients and supports the current 50% growth endpoint for susceptibility testing. However, it is still unclear if the gradual loss of efficacy observed for moderately trailing isolates in the larva model may be a reason for concern in selected vulnerable patient populations.

Update from a 12-Year Nationwide Fungemia Surveillance: Increasing Intrinsic and Acquired Resistance Causes Concern.

New data from the years 2012 to 2015 from the Danish National Fungemia Surveillance are reported, and epidemiological trends are investigated in a 12-year perspective (2004 to 2015). During 2012 to 2015, 1,900 of 1,939 (98%) fungal bloodstream isolates were included. The average incidence was 8.4/100,000 inhabitants, and this appears to represent a stabilizing trend after the increase to 10.1/100,000 in 2011. The incidence was higher in males than females (10.0 versus 6.8) and in patients above 50 years, and those changes were mainly driven by an increasing incidence among 80-to-89-year-old males (65.3/100,000 in 2014 to 2015). The proportion of Candida albicans isolates decreased from 2004 to 2015 (64.4% to 42.4%) in parallel with a doubling of the proportion of Candida glabrata isolates (16.5% to 34.6%, P < 0.0001). C. glabrata was more common among females (34.0% versus 30.4% in males). Following an increase in 2004 to 2011, the annual drug use stabilized during the last 2 to 3 years of that time period but remained higher than in other Nordic countries. This was particularly true for the fluconazole and itraconazole use in the primary health care sector, which exceeded the combined national levels of use of these compounds in each of the other Nordic countries. Fluconazole susceptibility decreased (68.5%, 65.2%, and 60.6% in 2004 to 2007, 2008 to 2011, and 2012 to 2015, respectively, P < 0.0001), and echinocandin resistance emerged in Candida (0%, 0.6%, and 1.7%, respectively, P < 0.001). Amphotericin B susceptibility remained high (98.7%). Among 16 (2.7%) echinocandin-resistant C. glabrata isolates (2012 to 2015), 13 harbored FKS mutations and 5 (31%) were multidrug resistant. The epidemiological changes and the increased incidence of intrinsic and acquired resistance emphasize the importance of continued surveillance and of strengthened focus on antifungal stewardship.

Evaluation of the in vitro activity of isavuconazole and comparator voriconazole against 2635 contemporary clinical Candida and Aspergillus isolates.

OBJECTIVE: The in vitro activity of isavuconazole was determined for 1677 Candida and 958 Aspergillus isolates from 2012 to 2014 with voriconazole as comparator. METHODS: Aspergillus isolates were screened for resistance using azole-agar. Aspergillus isolates that screened positive and all Candida isolates underwent EUCAST broth microdilution testing. Isolates were categorized as wild-type (wt) or non-wt, adopting EUCAST epidemiological cut-off values (ECOFFs) (where available) or wt upper limits (wtULs; two two-fold dilutions above the MIC50). The CYP51A gene was sequenced for non-wt Aspergillus fumigatus isolates. Itraconazole and posaconazole MICs were determined for selected Aspergillus isolates with isavuconazole MIC ≥2 mg/L. RESULTS: Isavuconazole MIC50 (range) (mg/L) against Candida species were: Candida albicans: ≤0.03 (≤0.03 to >4), Candida dubliniensis: ≤0.03 (≤0.03), Candida glabrata: ≤0.03 (≤0.03-4), Candida krusei: 0.06 (≤0.03-0.5), Candida parapsilosis: ≤0.03 (≤0.03-0.06), Candida tropicalis: ≤0.03 (≤0.03 to >4), Saccharomyces cerevisiae (anamorph: Candida robusta): ≤0.03 (≤0.03-0.5). Non-wt isavuconazole/voriconazole MICs were found for C. albicans: 0.8/1.0%, C. dubliniensis: 0/1.8%, C. glabrata: 14.9/9.5%, C. krusei: 2.7/1.4%, C. parapsilosis: 1.7/1.8%, C. tropicalis: 14.3/19.1% and S. cerevisiae: 10.0/0%. Isavuconazole MIC50 (range) (mg/L) against Aspergillus species were: A. fumigatus: 1 (≤0.125 to >16), Aspergillus niger: 2 (1-8), Aspergillus terreus: 1 (0.25-8), Aspergillus flavus: 1 (0.5-2), Aspergillus nidulans: ≤0.125 (≤0.125-0.25). Non-wt isavuconazole/voriconazole MICs were found for 13.7/15.2% A. fumigatus, 4.9/0% A. niger and 48.2/22.2% A. terreus. CONCLUSION: Isavuconazole displayed broad in vitro activity, similar to that of voriconazole. Up to 15% of C. glabrata, C. tropicalis and A. fumigatus isolates were non-wt, reflecting increased resistance at a reference centre and technical issues. Significant CYP51A alterations were reliably detected applying the isavuconazole breakpoint.

Azole-resistant Aspergillus fumigatus in Denmark: a laboratory-based study on resistance mechanisms and genotypes.

Azole-resistant Aspergillus fumigatus originating from the environment as well as induced during therapy are continuously emerging in Danish clinical settings. We performed a laboratory-based retrospective study (2010-2014) of azole resistance and genetic relationship of A. fumigatus at the national mycology reference laboratory of Denmark. A total of 1162 clinical and 133 environmental A. fumigatus isolates were identified by morphology, thermotolerance and/or ß-tubulin sequencing. Screening for azole resistance was carried out using azole agar, and resistant isolates were susceptibility tested by the EUCAST (European Committee on Antimicrobial Susceptibility Testing) E.Def 9.2 reference method and CYP51A sequenced. Genotyping was performed for outbreak investigation and, when appropriate, short tandem repeat Aspergillus fumigatus microsatellite assay. All 133 environmental A. fumigatus isolates were azole susceptible. However, from 2010 to 2014, there was an increasing prevalence of azole resistance (from 1.4 to 6% isolates (p <0.001) and 1.8 to 4% patients (p <0.05)) among the clinical isolates, with the well-known environmental CYP51A variant TR34/L98H responsible for >50% of the azole resistance mechanisms. Among 184 Danish A. fumigatus isolates, 120 unique genotypes were identified and compared to a collection of 1822 international genotypes. Seven (5.8%) Danish genotypes were shared between isolates within Denmark but with different origin, 19 (15.8%) were shared with foreign genotypes, and two (11.8%) of 17 genotypes of isolates carrying the TR34/L98H resistance mechanisms were identical to two Dutch TR34/L98H isolates. Our findings underlines the demand for correct identification and susceptibility testing of clinical mould isolates. Furthermore, although complex, genotyping supported the hypotheses regarding clonal expansion and the potential of a single origin for the TR34/L98H clone.

Posttreatment Antifungal Resistance among Colonizing Candida Isolates in Candidemia Patients: Results from a Systematic Multicenter Study.

The prevalence of intrinsic and acquired resistance among colonizing Candida isolates from patients after candidemia was investigated systematically in a 1-year nationwide study. Patients were treated at the discretion of the treating physician. Oral swabs were obtained after treatment. Species distributions and MIC data were investigated for blood and posttreatment oral isolates from patients exposed to either azoles or echinocandins for <7 or ≥ 7 days. Species identification was confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and internal transcribed spacer (ITS) sequencing, susceptibility was examined by EUCAST EDef 7.2 methodology, echinocandin resistance was examined by FKS sequencing, and genetic relatedness was examined by multilocus sequence typing (MLST). One hundred ninety-three episodes provided 205 blood and 220 oral isolates. MLST analysis demonstrated a genetic relationship for 90% of all paired blood and oral isolates. Patients exposed to azoles for ≥ 7 days (n = 93) had a significantly larger proportion of species intrinsically less susceptible to azoles (particularly Candida glabrata) among oral isolates than among initial blood isolates (36.6% versus 12.9%; P < 0.001). A similar shift toward species less susceptible to echinocandins among 85 patients exposed to echinocandins for ≥ 7 days was not observed (4.8% of oral isolates versus 3.2% of blood isolates; P > 0.5). Acquired resistance in Candida albicans was rare (<5%). However, acquired resistance to fluconazole (29.4%; P < 0.05) and anidulafungin (21.6%; P < 0.05) was common in C. glabrata isolates from patients exposed to either azoles or echinocandins. Our findings suggest that the colonizing mucosal microbiota may be an unrecognized reservoir of resistant Candida species, especially C. glabrata, following treatment for candidemia. The resistance rates were high, raising concern in general for patients exposed to antifungal drugs.

First detection of TR46/Y121F/T289A and TR34/L98H alterations in Aspergillus fumigatus isolates from azole-naive patients in Denmark despite negative findings in the environment.

Azole-resistant Aspergillus fumigatus harboring the TR34/L98H or TR46/Y121F/T289A alterations is increasingly found in Europe and Asia. Here, we present the first clinical cases of TR46/Y121/T289A and three cases of TR34/L98H outside the cystic fibrosis (CF) population in Denmark and the results of environmental surveys. Four patients (2012 to 2014) with 11 A. fumigatus and 4 Rhizomucor pusillus isolates and 239 soil samples (spring 2010 and autumn 2013, respectively) with a total of 113 A. fumigatus isolates were examined. Aspergillus isolates were screened for azole resistance using azole-containing agar. Confirmatory susceptibility testing was done using the EUCAST microbroth dilution EDEF 9.1 reference method. For relevant A. fumigatus isolates, CYP51A sequencing and microsatellite genotyping were performed. Three patients harbored TR34/L98H isolates. Two were azole naive at the time of acquisition and two were coinfected with wild-type A. fumigatus or R. pusillus isolates, complicating and delaying diagnosis. The TR46/Y121F/T289A strain was isolated in 2014 from a lung transplant patient. Genotyping indicated that susceptible and resistant Aspergillus isolates were unrelated and that no transmission between patients occurred. Azole resistance was not detected in any of the 113 soil isolates. TR34/L98H and TR46/Y121F/T289A alterations appear to be emerging in the clinical setting in Denmark and now involve azole-naive patients. Two recent soil-sampling surveys in Denmark were unable to indicate any increased prevalence of azole-resistant A. fumigatus in the environment. These findings further support the demand for real-time susceptibility testing of all clinically relevant isolates and for studies investigating the seasonal variation and ecological niches for azole-resistant environmental A. fumigatus.

Interleukin-28B polymorphisms are associated with hepatitis C virus clearance and viral load in a HIV-1-infected cohort.

Twenty-five per cent of individuals infected with hepatitis C virus (HCV) are able to clear HCV spontaneously. Differences in host genetics are believed to affect the outcome of HCV infection. We analysed an exonic, a promoter and an intronic single nucleotide polymorphism (SNP) of the interferon-λ3 coding interleukin (IL)-28B gene to study the relationship between IL28B SNPs and outcome of HCV infection. Among 206 HIV-1-infected Europeans with evidence of HCV infection, 47 (23%) individuals had cleared HCV and 159 (77%) had developed chronic infection. The exonic rs8103142 CT, the promoter rs12979860 CT and the intronic rs11881222 AG genotypes were associated with a decreased HCV clearance rate with adjusted odds ratios (aOR) of 0.3 (95% CI, 0.1-0.7), 0.4 (95% CI, 0.2-0.8) and 0.4 (95% CI, 0.2-0.8), respectively. The haplotype block TCG CTA was associated with a decreased HCV clearance rate (aOR 0.4, 95% CI, 0.2-0.8). Further, we found significant differences in HCV RNA levels among individuals chronically infected with HCV genotype 1 for rs8103142 and rs12979860 (P ≤ 0.05). Chronically infected individuals with HCV genotype 3 and with the favourable haplotype block CTA CTA had higher median HCV RNA levels than individuals with unfavourable haplotype blocks (P ≤ 0.05). Our findings suggest that IL28B may account for some differences in HCV outcome but that other factors including the viral genotype, host genetics and the host-virus interaction are likely to influence the outcome of HCV infection.

[Low-energy laser therapy in medial tibial stress syndrome]. / Lavenergi-laserbehandling af medialt tibialt stress-syndrom.

The effect of low-energy laser therapy on shin splints was examined in a randomized study with an unblinded design. Constripts from the Jutland Dragoon regiment with shin splints were given either active laser treatment (40 mW in 60 sec per cm tender tibia edge) or placebo laser. All patients were exempted from normal duty concerning activities like running and march. Forty-nine patients participated in the study, 23 in the laser group and 26 in the control group. From the start the study was designed to be double-blind, but by accident the code was broken towards the end of the study. We found no significant differences between the groups regarding pain visual analog score and readiness to return to active duty after 14 days.

[Diagnostic activity in general practice. 3. Diagnosis of breast diseases]. / Diagnostisk aktivitet i almen praksis. 3. Mammadiagnostisk aktivitet.

A random sample of 483 women from five practices representing a basic population of 4,867 women over the age of 35 years was selected in order to assess the examination activity for cancer of the breast. The examination activity in practice decreased with increasing age simultaneously with increase in the incidence of cancer of the breast. The possibility for a 93% total screening per five years is present but does not appear to be practically feasible. All of the positive findings occur in women seeking advice on account of symptoms. Instruction in self-examination will probably result in an increased number of referrals for this reason. Among the possibilities for teaching self-examination and regular control, women receiving hormonal contraception and women receiving substitution therapy at the menopause are suggested as possible clienteles. In both of these cases, annual examination of the breast should be performed in advance.

[Diagnostic activity in general practice. 2. Frequency of examination of urinary glucose and blood pressure determination among patients in general practice]. / Diagnostisk aktivitet i almen praksis. 2. Hyppigheden af urinundersøgelse for glukose og blodtryksmåling blandt praktiserende laegers patienter.

In order to estimate how frequently examinations of urine for glucosuria and blood pressure measurements are performed in the primary health care sector to detect common incapacitating diseases such as diabetes and hypertension, the authors carried out a study in five general practices in Denmark for a period of five years. The study was retrospective because it is not possible to study one's own behaviour prospectively. Nearly 1,000 persons aged 30 years or more were involved. 9% of the group had no contact with the health care sector during the period. In 49%, the urine had been tested at least once for glucose; 58% where women were concerned but only 40% of the men. Men under the age of 55 years were significantly underrepresented. Examination of the urine was only undertaken in 51% of the persons aged 55-74 years. Twelve new cases of diabetes were discovered, nine of these were diagnosed in general practice. Hypertension had been diagnosed in 52 patients before the registration period. 60% of the remainder had blood pressure measurements on at least one occasion during the period. Men under 55 years were significantly underrepresented (42%). Sixty-four new cases of hypertension were discovered, 59 of these in general practice. Screening for common diseases such as these is primarily performed by general practitioner. Several other works from general practice where no special efforts were made to systematize screening for diabetes or hypertension did not reveal any better results. It is estimated that 25% of diabetic patients and 50% of hypertensive patients remain undiagnosed. The present rather random screening for these diseases is insufficient.(ABSTRACT TRUNCATED AT 250 WORDS)

[Diagnostic activity in general practice. 1. Patients' contact patterns in rural and urban general practices]. / Diagnostisk aktivitet i almen praksis. 1. Patienternes kontaktmønster i almen by- og/eller landpraksis.

Contact with patients was described retrospectively for a period of five years by means of careful notes from the case records in five general practices in urban and rural areas chosen at random as a representative spot test. A total of 968 persons in group 1 of the Danish National Health Insurance (98-99% of the population), 485 men and 483 women or 9.7% of the group 1 insured persons over the age of 30 years had 10,410 consultations. This corresponds to 2.15 consultations per annum for persons in group 1 insurance. 91% of the patients visited the consultation. 17% of all the patient, twice as many women as men, were responsible for 50% of all the consultations. Women had 45% more consultations than men. Women in the age group 35-54 years constituted the group where most persons of the group visited general practice and this group had also the greatest total number of consultations, viz 30% of the total. Significantly more women than men had many consultations in this age group while significantly more men than women had only few consultations in the age group between 35-44 years. Expressed in percentages, the age group 55-64 years showed greatest similarity between men and women. Half of the men and slightly more than one third of all the women consulted their practitioners once per annum or more rarely. Patients of 75 years or over consulted their general practitioners most frequently.

Pleural empyema in a newborn infant. An unusual cause of respiratory distress.

In a 2-day-old baby with symptoms of respiratory distress a right-sided pleural empyema caused by haemolytic streptococci was found. Possible sources of infection are discussed. The mother had purulent discharge from a tooth a few weeks before delivery, and this may have been the focus. The baby was treated with pleural drainage and ampicillin with good result.

Auscultatory measurement of blood pressure performed by the doctor on duty.

A review is presented of the measurements of blood pressure on admission of 600 patients to the wards in a department of general medicine. Three doctors each examined 200 patients, distributed over two periods with an interval of one year. Large differences were found between the doctors as regards the distribution of the BPs, particularly the diastolic pressures. On the other hand, no differences were observed in the distributions during the two periods as far as the individual doctors were concerned. Great preference was shown for zero as the terminal digit and also for certain BP values. It is concluded that the observer introduces a considerable subjective factor in measuring the BP by auscultation. Correct performance of this simple procedure requires meticulous instruction.

Mortality from acute myocardial infarction before and after establishment of a coronary care unit.

A retrospective study of the mortality rate of acute myocardial infarction in two groups of patients treated before and after a coronary care unit was established showed no difference between them. Though it is difficult to compare two series retrospectively so far there are no well controlled studies to demonstrate clearly the value of coronary care units.