A prospective randomized controlled trial comparing PCR-based and empirical treatment with liposomal amphotericin B in patients after allo-SCT.

We compared the efficacy and safety of empirical plus PCR-based vs empirical liposomal amphotericin B treatment after Allo-SCT. Allo-SCT recipients were randomized to receive either PCR-based preemptive therapy (group A; n=198) or empirical antifungal therapy (group B; n=211) with liposomal amphotericin B. In group A, therapy was started after one positive PCR result or after 120 h of febrile neutropenia refractory to broad-spectrum antibacterial therapy. In group B, liposomal amphotericin B was started after 120 h of refractory febrile neutropenia. Demographic and clinical characteristics were well balanced. A total of 112 (57.1%) patients in group A and 76 (36.7%) patients in group B received antifungal therapy (P<0.0001). Twelve patients in group A and 16 patients in group B developed proven invasive fungal infection (IFI). Survival curves showed better survival until day 30 when close PCR monitoring was performed (mortality 1.5 vs 6.3%; P=0.015), but there was no difference at day 100. At day 100, no difference was observed in the incidence of IFI (primary end point) and survival between the two arms. Further studies are required to assess the benefit of using PCR in patients after SCT.

Identification of medically important fungi by the Pyrosequencing technology.

The Pyrosequencing technology was used for identification of different clinically relevant fungi. The tests were performed on amplicons derived from the 18S rRNA gene using polymerase chain reaction (PCR) universal primers for amplification. Sequencing was performed up to 40 bases in a variable region with a designed general sequencing primer and the Pyrosequence data were analyzed by BLAST sequence search in the GenBank database. DNA from a total of 21 fungal specimens consisting of nine strains of clinically relevant fungi and 12 clinical specimens from patients suffering from proven invasive fungal infections were PCR-amplified and analyzed by gel electrophoresis, PCR-enzyme-linked immunosorbent assay (ELISA) and the Pyrosequencing technology. All data obtained by the Pyrosequencing technology were in agreement with the results obtained by PCR-ELISA using species/genus-specific oligonucleotides and were as well in accordance with the culture results. The results demonstrate that the Pyrosequencing method is a reproducible and reliable technique for identification of fungal pathogens.

Liposomal amphotericin B (AmBisome) for fungal infections in immunocompromised adults and children.

Invasive fungal infections are rare but life-threatening infections, most often occurring in immunocompromised patients. For a long time, Amphotericin B has been the best choice for treatment, because it is fungicidal with a broad antifungal spectrum and minimal risk of resistance development. The therapeutic use of amphotericin B has, however, been limited by its toxicity-both acute as well as chronic. To counter this, amphotericin B has been encapsulated in liposomes, which reduces its toxicity and allows higher doses to be given. Ambisome is a true, spherical, small unilamellar liposome with a median size of 80 nm. The pharmacokinetic profile was changed, and the maximum concentration and AUC of amphotericin B after AmBisome treatment were greater than those found with the conventional drug. The highest tissue concentrations of AmBisome were found in the liver and spleen, and less than 1% of the administered dose was recovered in other organs. At Huddinge University Hospital, we were the first to use and report on the experience of AmBisome. We now have more than 12 years' experience in transplant recipients, with a good safety profile, improved rate of curing mycological proven infections and reduced mortality in fungal infections. In two placebo-controlled prophylactic trials, we found that AmBisome was effective for preventing fungal colonization and invasive fungal infections, respectively, in allogeneic stem cell and liver transplantation. In uncontrolled and, more recently, in randomized controlled studies at other centers, AmBisome has revealed less toxicity and an efficacy equal or superior to that of the conventional drug in treating neutropenia-associated fever and proven invasive fungal infections in both adults as well as in children. Although investigators tend to increase the dose used, the optimal dose for probable or proven infection is still under debate. Based on our own experience in using AmBisome and the experience at other centers, we can conclude that AmBisome represents a major breakthrough in the treatment of invasive fungal infections, especially in immunocompromised patients.

Treatment of severe acute graft-versus-host disease with anti-thymocyte globulin.

Severe acute graft-versus-host disease (GVHD) is one of the major complications after haematopoietic stem-cell transplantation (HSCT). Treatment of severe GVHD is difficult and the condition is often fatal. One proposed method of improving the therapy is to include anti-thymocyte globulin (ATG). Here, we will report our results in 29 patients using ATG as part of treatment for severe steroid-resistant acute GVHD. Four patients suffered from grade II, 13 from grade III and 12 from grade IV GVHD. Median time to grade II GVHD was 24 d (range 7-91 d) and to grade III was 29 d (range 8-55 d) after HSCT. Five different ATG preparations were used, rabbit ATG (R-ATG), BMA 031, OKT3, ATG-Fresenius and Thymoglobuline. All patients had skin involvement, 26 also had gut involvement and 25 had liver involvement. The rate of response to treatment was best in skin involvement (72%), while liver and gut involvement showed lower response rates (38%). Eleven patients survived more than 90 d, 7 of them developed chronic GVHD, 1 developed mild GVHD, 1 developed moderate GVHD and 5 developed severe GVHD. Survival at 100 d was 37% and at 1 yr it was 12%. Most patients died of GVHD, with virus or fungal infections as contributing causes of death. To conclude, treatment of severe acute GVHD is difficult and ATG, in our hands, adds nothing to conventional pharmacological treatment.

Need for alternative trial designs and evaluation strategies for therapeutic studies of invasive mycoses.

Studies of invasive fungal infections have been and remain difficult to implement. Randomized clinical trials of fungal infections are especially slow and expensive to perform because it is difficult to identify eligible patients in a timely fashion, to prove the presence of the fungal infection in an unequivocal fashion, and to evaluate outcome in a convincing fashion. Because of these challenges, licensing decisions for antifungal agents have to date depended heavily on historical control comparisons and secondary advantages of the new agent. Although the availability of newer and potentially more effective agents makes these approaches less desirable, the fundamental difficulties of trials of invasive fungal infections have not changed. Therefore, there is a need for alternative trial designs and evaluation strategies for therapeutic studies of invasive mycoses, and this article summarizes the possible strategies in this area.

Pharmacokinetic interaction between single oral doses of diltiazem and sirolimus in healthy volunteers.

AIM AND BACKGROUND: The pharmacokinetic interaction between sirolimus, a macrolide immunosuppressant metabolized by CYP3A4, and the calcium channel blocker diltiazem was studied in 18 healthy subjects. Several clinically important interactions have previously been reported for other immunosuppressive drugs that are metabolized by the same enzyme and for calcium antagonists. METHODS: Healthy subjects who were 20 to 43 years old participated in an open, three-period, randomized, crossover study of the pharmacokinetics of a single 10-mg oral dose of sirolimus, a single oral 120-mg dose of diltiazem, and the two drugs given together. The three study periods were separated by a 21-day washout phase. RESULTS: The geometric mean (90% confidence interval) whole blood sirolimus area under the plasma concentration time-curve increased 60% (35%-90%), from 736 to 1178 ng x h/mL, and maximum concentration increased 43% (14%-81%), from 67 to 96 ng/mL, with diltiazem coadministration, whereas the mean elimination half-life of sirolimus decreased slightly, from 79 to 67 hours. Apparent oral clearance and volume of distribution of sirolimus decreased with 38% and 45%, respectively, when sirolimus was given with diltiazem. The plasma maximum concentration and area under the plasma concentration-time curve of diltiazem, desacetyldiltiazem, and desmethyldiltiazem were unchanged after coadministration of sirolimus, and no potentiation of the effects of diltiazem on diastolic or systolic blood pressure or on the electrocardiographic parameters was seen. CONCLUSIONS: Single-dose diltiazem coadministration leads to higher sirolimus exposure, presumably by inhibition of the first-pass metabolism of sirolimus. Because of the pronounced intersubject variability in the extent of the sirolimus-diltiazem interaction, whole blood sirolimus concentrations should be monitored closely in patients treated with the two drugs.

Combined pancreas and kidney transplantation improves survival in patients with end-stage diabetic nephropathy.

The purpose of this study was to find out whether prolonged normoglycemia, as achieved by a successful pancreas transplantation, can improve survival in patients with insulin-dependent diabetes mellitus. A retrospective analysis of actual 10-yr patient survival rates was done for all renal graft recipients who were given transplants more than 10 yr ago but within the cyclosporin era (i.e. 1981-1988). The actual 10-yr patient survival rate in non-diabetic renal graft recipients was 72%, In recipients of pancreas and kidney grafts and with prolonged function of the pancreas graft, the survival rate was 60%, whereas in patients subjected to simultaneous pancreas and kidney transplantation, but where the pancreatic grafts failed within 2 yr, the survival rate was 33%. In diabetic recipients of kidney transplants alone, the survival rate was 37%. The patient survival rate was substantially higher in non-diabetic patients and patients with functioning pancreas grafts compared with diabetic patients with kidney transplants alone or with failed pancreas grafts. We speculate that the decrease in mortality was due to the beneficial effect of long-term normoglycemia on diabetic late complications.

Identification of rare Candida species and other yeasts by polymerase chain reaction and slot blot hybridization.

Invasive candidiasis has become a major cause of morbidity and mortality in immunocompromised hosts. Here we describe a fast and reliable DNA extraction and PCR amplification method in combination with a slot blot hybridization assay. A genus-specific probe was designed that allowed to detect DNA from a broad range of Candida species and 3 other yeasts. In addition, species-specific oligonucleotides for emerging Candida and other yeast species allowed to identify DNA extracted from Candida lusitaniae, Candida humicola, Candida kefyr, Candida inconspicua, Candida solani, Malassezia furfur and Trichosporon cutaneum. A sensitivity of at least 10(1) CFU, corresponding to 100 fg of fungal DNA, was documented for all species-specific probes and the common Candida probe. In addition, the 18S rRNA genes of 7 yeast species (C. humicola, C. kefyr, C. solani, C. inconspicua, C. norvegensis, C. utilis and M. furfur) were completely sequenced. The sequencing primers described bind to highly conserved primer binding sites. Therefore, these primers would allow rapid cycle sequence of additional ribosomal genes throughout the whole kingdom of fungi.

Antifungal compounds: controversies, queries and conclusions.

A new session in this Congress was the development of a discussion panel regarding controversies and queries about the main topics of treatment and prophylaxis of severe systemic mycoses. Experts presenting each side of three controversial areas provided an interchange of ideas and clarified those areas where there remain substantial disagreements. Some common recommendations have been made, and some differences persist. The contents of this session are compiled in this paper. Due to the limitations of space, this paper presents the most relevant parts of each presentation. The large and up-to-date list of references should be useful to gain a better understanding of these subjects.

PCR and other non-culture methods for diagnosis of invasive Candida infections in allogeneic bone marrow and solid organ transplant recipients.

In this prospective study 197 serum and 152 urine samples were collected from 40 bone marrow and solid organ transplant recipients with clinically suspected invasive fungal infection before, during and after empirical treatment with lipid formulation of amphotericin B or fluconazole. Serum was analysed by Candida polymerase chain reaction (PCR) and urine by measurement of D/L-arabinitol ratio. One serum from each patient was also tested for concentration of (1-->3)-beta-glucan and two commercial Candida antigens. Invasive fungal infection was diagnosed in four candidosis and one aspergillosis patients (13%). Positive PCR, elevated D/L-arabinitol ratio, (1-->3)-beta-glucan concentration and antigens were detected in nine, 15, 17, and seven patients, respectively. The agreement between PCR and D/L-arabinitol assays was poor. However, 56% agreement was observed between positive PCR and beta-glucan and/or antigen assays, and 60% agreement between positive D/L-arabinitol and beta-glucan and/or antigen assays. Combination of several non-culture assays is needed to diagnose invasive fungal infection in high-risk transplant recipients. No single test was sufficient for diagnosis.

Fungal prophylaxis by reduction of fungal colonization by oral administration of bovine anti-Candida antibodies in bone marrow transplant recipients.

Candida overgrowth and invasion constitute a serious threat with a high mortality in BMT recipients. Currently available topical antifungal prophylaxis is largely ineffective, and as resistance to existing, absorbable drugs for systemic use is rapidly developing, new forms of therapy are needed. We investigated the effect of oral treatment of BMT recipients with a bovine immunoglobulin product derived from animals immunized against several Candida species. The natural Candida colonization was first followed in 19 patients to establish the colonization pattern. Half of the patients were found to be colonized prior to transplantation and altogether 72% were colonized at some point during follow-up. Those with a high pre-transplant concentration of Candida in saliva (>100 CFU/ml) remained colonized throughout the BMT treatment period. The therapeutic effect was monitored in two other patient groups. The first group consisted of nine patients, where, due to a low number of primary colonized patients, response in colonized patients was suggestive of a therapeutic effect. In the second group, 10 patients with a high level of colonization (>100 CFU/ml) were given 10 g daily of the product in three divided doses. The results suggest a treatment-related reduction in Candida colonization in a majority (7/10) of patients and one patient became completely negative. As no adverse effects were noted, our findings encourage additional studies in immunocompromised, transplant patients.

Prophylaxis against fungal infections in transplant recipients: possible approaches.

Although the morbidity and mortality associated with invasive fungal infections in transplant recipients is high, the optimal approach to antifungal prophylaxis is controversial. Most fungal infections occur shortly after the trans- plantation during maximum immunosuppression and are caused by Candida or Aspergillus spp. Nonspecific strategies for prevention do not differ from those used in other patients at risk. They consist mainly of a reduction in risk factors, such as removal of plants from around the patient, separation of the patient from the vicinity of construction sites or improvement in hospital care by isolation and careful nursing of the patient, with strict hygiene. A more controversial issue is primary antifungal chemoprophylaxis, since there are few well designed trials of this intervention, and most of the patients studied have had haematological diseases. Orally administered antifungal drugs that are not absorbed through the gastrointestinal tract have not shown any evidence of a prophylactic effect to date. Controlled trials of systemically administered or orally absorbed drugs in specific transplant recipients have, however, proved effective. In allogeneic and autologous bone marrow transplants, fluconazole 400 mg/day was effective when administered from the conditioning treatment period through the neutropenic period. In liver transplant recipients, either fluconazole 400 mg/day for 10 weeks or liposomal amphotericin B 1 mg/kg/day for 5 days significantly reduced the incidence of invasive fungal infections. However, one must be aware of the risk of fluconazole-resistant fungi and the possibility of selection. In patients with a history of previous fungal infection, secondary prophylaxis may be of value, although data are limited. For recipients of transplants other than bone marrow or liver, there are insufficient data to recommend general prophylaxis.

Urinary tract infections following renal transplantation.

The incidence of urinary tract infections (UTIs) in 363 adult renal transplant recipients transplanted during the period 1990-96 has been analysed. UTI occurred in 96 patients (26%), most frequently during the first year after transplantation. Female recipients had significantly more UTI than male recipients (49% vs. 14%, p < 0.0001). There was no difference in the incidence of UTI between recipients receiving pig-tail catheters as ureteral stents or not, the figures being 21% vs. 28%, respectively. Age had no influence on the incidence of UTI. In 341 patients treated with cyclosporine the incidence of UTI was 28%, while in 15 patients treated with FK-506 only 1 patient (7%) had a UTI (ns). The majority of organisms cultured were gram-negative (76%), with approximately 1/3 being Escherichia coli and 1/5 being Enterococcus and Klebsiella/Enterobacter. The bacterial spectrum was not influenced by the recipient's age. UTI had no effect on the number of rejections, or on graft and patient survival in living donor transplant recipients. No significant difference was found in graft and patient survival rates at 3 yr between patients who had UTI or no UTI.

Deep Candida infection in children with leukaemia: clinical presentations, diagnosis and outcome.

OBJECTIVES: To analyse the clinical features associated with deep Candida infection (DCI) and the outcome in children with leukaemia, and to evaluate various diagnostic methods. MATERIALS AND METHODS: Serum samples were analysed to determine Candida IgA, IgM and IgG antibodies and defect free C. albicans glucoprotein antigen and C. enolase antigen in eight children who had nine episodes of DCI and six with suspected DCI. RESULTS: DCI occurred shortly after the leukaemia diagnosis (median 40 days) or after the leukaemia relapse (median 30 days). Children with DCI had fever (100%), skin lesions/exanthema (45%), oral thrush (45%), oesophagitis (22%) and laryngo-tracheitis (22%). Candida endocarditis, arthritis and hepatic candidosis were diagnosed in one patient each. Two children with disseminated candidosis died in leukaemia relapse. In patients with C. albicans infections serology had a sensitivity of 83%. However, in patients with C. parapsilosis infection antibody detection was negative. As the patients were cured of their Candida infection, the IgG antibodies disappeared and the IgM and IgA antibodies fell within the normal range for age. CONCLUSION: DCI in children occurs shortly after the leukaemia diagnosis or shortly after relapse of leukaemia. The clinical features are many. Candida serology may help to diagnose or confirm DCI. The dynamics of antibody titres may help to establish the efficacy of antifungal treatment.