Disseminated histoplasmosis and AIDS: a prospective and multicentre study to evaluate the performance of different diagnostic tests.

The burden of histoplasmosis has been poorly documented in most of the endemic areas for the disease, including Brazil. Also, modern non-culture-based diagnostic tests are often non-available in these regions. This was a prospective cohort study in HIV-infected patients with suspected disseminated disease evaluated with different diagnostic tests. Patients were enrolled in three referral medical centres in Porto Alegre, Brazil. Among 78 evaluated patients, disseminated histoplasmosis was confirmed in eight individuals (10.3%) by the means of classical (culture/histopathology) tests. Antigen detection in the urine was found to be more sensitive: IMMY® ALPHA ELISA detected 13 positive cases (16.7%) and the in-house ELISA test developed by the Centers for Disease Prevention and Control (CDC) detected 14 (17.9%). IMMY® and CDC tests provided concordant results in 96.2% of cases. This is the first study to compare the performance of the in-house CDC ELISA test with the IMMY® commercial test for the diagnosis of histoplasmosis, and a high degree of concordance was observed. The study revealed that H. capsulatum is an important agent of disseminated disease in AIDS patients in Brazil, reinforcing the importance of making available modern diagnostic tests as well as safer antifungal agents for the treatment of histoplasmosis.

Correlation between the presence of degenerated inclusion-bearing cells in voided urine samples and the occurrence of polyomavirus infection.

OBJECTIVE: The purpose of the present, prospective, cohort study was to monitor urine cytology samples from recipients of renal transplants to search for the occurrence of decoy cells and degenerated inclusion-bearing cells with an aim to correlate the existence of these cells with molecular detection of polyomavirus BK (BKV) DNA in urine. MATERIAL AND METHODS: This study included patients who underwent renal transplantation. Patients had their urine tested quarterly, during the first year post-transplantation, for the presence of decoy cells and degenerated cells, as well as by quantitative determination of BKV load in the urine and plasma. RESULTS: Three hundred and sixty-one examinations were performed on 101 patients within 12 months of attendance. Urine cytology results were: 198 (54.9%) negative and 60 (16.6%) positive for the presence of viral cytopathic effects depending on the presence of BKV infection, 72 (19.9%) positive for the manifestation of degenerated cells and 31 (8.6%) unsatisfactory for analysis. There was a subtle tendency towards the presence of degenerated inclusion-bearing cells in cases in which the virus was detected in voided urine. However, the presence of degenerated cells exhibited a tendency to BKV positivity in months 3, 6 and 9 and, exclusively in month 12, this trend was statistically significant. CONCLUSIONS: There were not enough strong morphological and staining elements to state the origin of the degenerated cells or to describe the nature of the infection (viral or bacterial), given that these cells were undergoing an apoptotic process in post renal transplant patients.

Thrombin-activatable Fibrinolysis Inhibitor (TAFI) as a Novel Prognostic Factor After Orthotropic Liver Transplantation: A Pilot Study.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI), a liver-produced coagulation factor, has been associated with higher mortality in cirrhotic patients, but there has not been any description of its role in perioperative care in liver transplantation cases. METHODS: A total of 21 patients were included. Serum TAFI levels were determined at 3 time points: preoperatively (TAFI pre), immediately postoperative (TAFI PO), and 24 hours postoperatively (TAFI 24 h). The main outcome was the physiological pattern of TAFI in the perioperative period of liver transplantation. The secondary outcomes were the association between TAFI and early allograft dysfunction (EAD) as well as that of TAFI and 6-month mortality. RESULTS: TAFI levels increased at the 24-hour time point, compared to the other 2 time points (TAFI pre, P = .007; TAFI PO, P = .0001). Early allograft dysfunction occurred in 2 of 21 patients, both demonstrating lower TAFI 24 h levels compared to those who did not develop this complication (3.0 ± 0.2 vs 1.5 ± 0.3; P = .0001). Three patients who died all demonstrated lower levels of TAFI pre (1.3 ± 0.1 vs 2.5 ± 0.5; P = .001) and TAFI PO (1.2 ± 0.1 vs 2.4 ± 0.4; P = .001) compared to the survivors. CONCLUSIONS: These findings suggest that the determination of TAFI levels-both pre- and postoperatively-may be of clinical relevance in liver transplant recipients.

Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus.

To investigate azole resistance in clinical Aspergillus isolates, we conducted prospective multicenter international surveillance. A total of 3,788 Aspergillus isolates were screened in 22 centers from 19 countries. Azole-resistant A. fumigatus was more frequently found (3.2% prevalence) than previously acknowledged, causing resistant invasive and noninvasive aspergillosis and severely compromising clinical use of azoles.

Risk factors for hepatotoxicity in solid organ transplants recipients being treated for tuberculosis.

BACKGROUND: Tuberculosis (TB) is associated with high morbidity and mortality in solid organ transplant (SOT) recipients. Also, SOT patients have a 20- to 74-fold increase in the chance of developing TB compared to the general population. Here we evaluated the incidence of hepatotoxicity in SOT recipients on treatment for TB and determined risk factors for liver toxicity in these patients. PATIENTS AND METHODS: Retrospective cohort conducted in a reference hospital for SOT in Southern Brazil. All SOT recipients who underwent TB treatment during the years 2000-2012 were considered for the study. RESULTS: A total of 69 patients were included in the study and 23 had liver toxicity (incidence 33.3%). Independent risk factors for hepatotoxicity were rifampin use at doses of ≥600 mg daily (P = .016; OR 2.47; 95% CI, 1.18-5.15) and lung transplantation (P = .017; OR 2.05; 95% CI, 1.14-3.70). Kidney transplantation appeared as a protective factor (P = .036; OR 0.50; 95% CI, 0.26-0.96). Mortality was higher in the patients who had hepatotoxicity (43.5%), compared with those who did not (19.6%). CONCLUSION: In this study, the use of rifampin at doses of 600 mg daily or higher was found to be an independent risk factor for liver toxicity in SOT recipients. The importance of additional risk factors for hepatotoxicity, such as lung transplantation as well as the protective role of kidney transplantation, should be better investigated in SOT recipients being treated for TB.

Fungi in the respiratory tract of horses with recurrent airway obstruction / Presença de fungos no trato respiratório de equinos e sua associação com obstrução recorrente das vias aéreas

This study aims to compare the concentration of viable fungi, especially those of the genus Aspergillus in the respiratory tract of stabled horses with and without Recurrent Airway Obstruction (RAO). Thirty two housed horses from four equestrian centers in Brazil were included in the study. These animals were submitted to clinical examination and to a respiratory sample collection. They were categorized into two groups: healthy and RAO-affected horses. Samples obtained by tracheobronchial washes were evaluated for fungal microscopy, quantitative culture and Aspergillus spp. quantification. Eighteen healthy and 14 RAO-affected horses were studied. Fungi were more frequently recovered in the RAO group, in comparison to controls, for both fungal microscopy (P<0.0001), fungal culture (P<0.0001) and Aspergillus spp. quantitative culture (p=0.001). In conclusion, horses with RAO have significantly higher fungal load in the respiratory tract in comparison to healthy horses. The implications of these findings in terms of the pathogenesis of RAO deserve additional investigation...

Este estudo objetivou comparar a concentração de fungos viáveis, especialmente do gênero Aspergillus, no trato respiratório de equinos estabulados com e sem obstrução recorrente das vias aéreas (ORVA). Trinta e dois equinos provenientes de quatro centros de treinamento equestre do Brasil foram incluídos no estudo. Os animais foram submetidos a exame clínico e coleta de amostra respiratória, sendo categorizados em dois grupos: sadios e ORVA. Os lavados traqueobrônquicos obtidos foram avaliados por exame micológico direto e cultivo quantitativo. Ao todo, 18 equinos saudáveis e 14 com ORVA foram estudados. Fungos foram mais frequentemente detectados em amostras do grupo ORVA em comparação com o grupo controle, tanto no exame micológico direto (P<0,0001) e cultivo quantitativo (P<0,0001) quanto na concentração de unidades formadoras de colônias (UFC) de Aspergillus spp. isolada em cultivo (p=0,001). Equinos com ORVA têm maior concentração de propágulos fúngicos no trato respiratório em comparação com animais sadios. As implicações desses achados na patogenia da ORVA merecem maior atenção e investigação...

Different risk factors for candidemia occur for Candida species belonging to the C. parapsilosis complex.

Previous studies have demonstrated reduced virulence in the species that comprise the Candida parapsilosis complex. We investigated a cohort of 93 patients with candidemia caused by this complex. Most infections were caused by C. parapsilosis (80.6%), followed by C. orthopsilosis (18.3%) and C. metapsilosis (1.1%). Renal failure (P < 0.001) and chronic liver diseases (P = 0.019) were more frequently encountered with infections caused by the C. orthopsilosis group, suggesting an association with patients who had a greater state of immune suppression in comparison with infections caused by C. parapsilosis sensu stricto.

Prolonged respiratory viral shedding in transplant patients.

Respiratory viral infections are frequent causes of morbidity in transplant patients. We screened symptomatic adult transplant recipients for respiratory viruses in a cohort of patients attending a referral medical center in Brazil. The duration of viral shedding and the prevalence of viral codetections were also determined. During a 1-year period (2011-2012), swabs were obtained from 50 patients. An in-house polymerase chain reaction panel designed to detect 10 viruses was used. Viruses were identified in 19 (38%) patients, particularly parainfluenza III (32%) and the respiratory syncytial virus (20%); multiple viruses were identified in 26% of patients. Prolonged viral shedding was observed with 60% of individuals excreting viruses for >10 days. The clinical and epidemiologic relevance of prolonged viral shedding remains to be determined.

Improvement in the outcome of invasive fusariosis in the last decade.

Invasive fusariosis (IF) has been associated with a poor prognosis. Although recent series have reported improved outcomes, the definition of optimal treatments remains controversial. The objective of this study was to evaluate changes in the outcome of IF. We retrospectively analysed 233 cases of IF from 11 countries, comparing demographics, clinical findings, treatment and outcome in two periods: 1985-2000 (period 1) and 2001-2011 (period 2). Most patients (92%) had haematological disease. Primary treatment with deoxycholate amphotericin B was more frequent in period 1 (63% vs. 30%, p <0.001), whereas voriconazole (32% vs. 2%, p <0.001) and combination therapies (18% vs. 1%, p <0.001) were more frequent in period 2. The 90-day probabilities of survival in periods 1 and 2 were 22% and 43%, respectively (p <0.001). In period 2, the 90-day probabilities of survival were 60% with voriconazole, 53% with a lipid formulation of amphotericin B, and 28% with deoxycholate amphotericin B (p 0.04). Variables associated with poor prognosis (death 90 days after the diagnosis of fusariosis) by multivariable analysis were: receipt of corticosteroids (hazard ratio (HR) 2.11, 95% CI 1.18-3.76, p 0.01), neutropenia at end of treatment (HR 2.70, 95% CI 1.57-4.65, p <0.001), and receipt of deoxycholate amphotericin B (HR 1.83, 95% CI 1.06-3.16, p 0.03). Treatment practices have changed over the last decade, with an increased use of voriconazole and combination therapies. There has been a 21% increase in survival rate in the last decade.

Major variations in Aspergillus fumigatus arising within aspergillomas in chronic pulmonary aspergillosis.

Aspergillomas develop from progressive layers of mycelial growth on the walls of pulmonary cavities over months. Aspergillomas are characteristic of chronic pulmonary aspergillosis and are a risk factor for azole resistance. We investigated genotypic and phenotypic alterations in Aspergillus fumigatus recovered from aspergillomas. Aspergillomas were removed from three patients (two at surgery, one at autopsy) and dissected. Overall 92 colonies of A. fumigatus were isolated. Microsatellite typing was conducted to determine genetic type. Itraconazole, voriconazole and posaconazole susceptibilities were performed. The cyp51A gene was sequenced in 22 isolates. Isolates from Patient 1 (n = 25) were azole susceptible and resistant, although all cyp51A sequences were wild type, the isolates split into two distinct clades. In Patient 2, isolates were less variable (n = 10), all were azole susceptible. In Patient 3 only azole-resistant strains (n = 57) were isolated, with M220K or M220T Cyp51A alterations, and microevolution was indicated. Marked diversity was observed in isolates from these patients; revealing differences in azole susceptibility, mechanism of resistance and genetic type. Importantly, routine sampling from respiratory specimens proved suboptimal in all cases; azole resistance was missed (Patient 1), cultures were negative (Patient 2) and high-level posaconazole resistance was not detected (Patient 3).

Fungal spore concentrations in two haematopoietic stem cell transplantation (HSCT) units containing distinct air control systems.

Invasive fungal diseases have emerged as important causes of morbidity and mortality in haematological patients. In this study air samples were collected in two haematopoietic stem cell transplantation (HSCT) units, in which distinct air-control systems were in place. In hospital 1 no high-efficiency particulate air (HEPA) filter was available whereas in hospital 2 HSCT rooms were equipped with HEPA filters, with positive air pressure in relation to the corridor. A total of 117 samples from rooms, toilets and corridors were obtained during December 2009 to January 2011, using a six-stage Andersen sampler. In both hospitals, the concentration of potentially pathogenic fungi in the air was reduced in patients' rooms compared to corridors (P < 0·0001). Despite the presence of a HEPA filter in hospital 2, rooms in both hospitals showed similar concentrations of potentially pathogenic fungi (P = 0·714). These findings may be explained by the implementation of additional protective measures in hospital 1, emphasizing the importance of such measures in protected environments.

Drinking yerba mate infusion: a potential risk factor for invasive fungal diseases?

Yerba mate (Ilex paraguariensis) infusion is a very popular drink in South America. Although several studies have evaluated the potential for fungal contamination in foodstuff, very few investigations have been conducted with yerba mate samples. In order to evaluate for the presence of potentially pathogenic fungi, here we studied 8 brands of yerba mate commercially available in Southern Brazil. Fungal survival in adverse conditions such as gastric pH was determined by incubating samples at pH 1.5. Because hot water is generally used to prepare yerba mate infusion, the effect of several temperatures on fungal growth was also investigated. All but 1 yerba mate brand showed substantial fungal growth, in the range of <10­4900 colony-forming units per gram. Some of these fungi were able to survive extreme variations in pH and temperature. Because of the potential for yerba mate to carry pathogenic fungi, immunocompromised patients may be at risk of acquiring invasive fungal diseases by drinking yerba mate infusion.

Azole resistance in allergic bronchopulmonary aspergillosis and Aspergillus bronchitis.

Oral azole antifungal therapy is used extensively for all forms of aspergillosis, including allergic bronchopulmonary aspergillosis (ABPA). However, long-term therapy may increase the risk of resistance. Here we report itraconazole and voriconazole resistance with reduced susceptibility to posaconazole in Aspergillus fumigatus in two patients exposed to itraconazole. Patients were diagnosed with ABPA and Aspergillus bronchitis related to innate immune defects. An azole susceptible strain was initially isolated from patient 1, but later a genetically different azole-resistant strain was cultured, possibly related to sub-therapeutic itraconazole levels, which could be a trigger for selection of resistance. The mechanism of resistance identified in this case was an L98H change in Cyp51A, accompanied by a tandem repeat in the promoter region of cyp51A leading to increased expression. No cyp51A mutation was found in azole-resistant isolates recovered from patient 2. Both patients responded to posaconazole, with plasma levels of >1.0 mg/L. Subsequently, susceptible strains of different molecular types were cultured from both patients, suggesting eradication and replacement.

Interlaboratory reproducibility of a microsatellite-based typing assay for Aspergillus fumigatus through the use of allelic ladders: proof of concept.

An interlaboratory study was performed with the aim of investigating the reproducibility of a multiplex microbial microsatellite-based typing assay for Aspergillus fumigatus in different settings using a variety of experimental and analytical conditions and with teams having variable prior microsatellite typing experience. In order to circumvent problems with exchange of sizing data, allelic ladders are introduced as a straightforward and universally applicable concept for standardization of such typing assays. Allelic ladders consist of mixtures of well-characterized reference fragments to act as reference points for the position in an electrophoretic trace of fragments with established repeat numbers. Five laboratories independently analysed six microsatellite markers in 18 samples that were provided either as DNA or as A. fumigatus conidia. Allelic data were reported as repeat numbers and as sizes in nucleotides. Without the use of allelic ladders, size differences of up to 6.7 nucleotides were observed, resulting in interpretation errors of up to two repeat units. Difficulties in interpretation were related to non-specific amplification products (which were resolved with explanation) and bleed-through of the different fluorescent labels. In contrast, after resolution of technical or interpretive problems, standardization of sizing data by using allelic ladders enabled all participants to produce identical typing data. The use of allelic ladders as a routine part of molecular typing using microsatellite markers provides robust results suitable for interlaboratory comparisons and for deposition in a global typing database.

Voriconazole plasma monitoring.

AIMS: Very little information is available regarding the use of voriconazole drug monitoring in children with invasive fungal infections. The purpose of this study was to report the cases of five paediatric patients treated with voriconazole, in which plasma levels were used to monitor therapy. METHODS: Five children treated with voriconazole were included in this case series. Voriconazole plasma levels were determined using either a bioassay or liquid chromatography-tandem mass spectrometry. RESULTS: The patients' ages ranged from 2 to 10 years old (mean 6.2 years). Three patients had acute leukaemia and two had suffered severe burn injuries. Doses administered varied from 3.4 mg/kg every 12 h to 8.1 mg/kg every 8 h. Plasma voriconazole concentrations were unpredictable for these paediatric patients. Subtherapeutic levels were frequently observed, despite progressive increments in dosage. For others, voriconazole levels markedly increased after a small increment in dosage. Phenobarbitone caused important drug interactions with voriconazole for one [corrected] of the patients. CONCLUSIONS: The dose administered did not correlate with exposure as measured by plasma levels of voriconazole. While the optimal dosage for voriconazole in children is still unknown, drug monitoring seems warranted to ensure adequate exposure, and after dose increments to prevent excessive exposure. Drug interactions significantly altered exposure.

New and emerging treatments for fungal infections.

Although several new antifungal drugs have been licensed in the last 5 years, some patients remain difficult to treat. The main reasons for this include intrinsic or acquired antifungal resistance, organ dysfunction preventing the use of some agents and drug interactions. In addition, some drugs penetrate poorly into sanctuary sites including eye and urine, and others are associated with considerable adverse events. Here, we review the preclinical and clinical development progress with four new antifungal agents: isavuconazole, ravuconazole, albaconazole and aminocandin. Isavuconazole and ravuconazole are extremely similar, with a broad spectrum of activity, a very long half-life and large volume of distribution and good in vivo data supporting their efficacy in invasive aspergillosis and candidosis. Both compounds are in early Phase 3 development. Albaconazole has also shown very potent activity against species of Candida, Cryptococcus and Aspergillus. It was well tolerated and effective in women with vaginal candidosis. Aminocandin is an intravenous-only echinocandin with in vivo activity against Candida spp. and Aspergillus spp. Its extended half-life probably permits dosing less frequently than once a day. Overall these new antifungal agents in development offer extended half-lives, possibly reduced drug interaction profiles and good tolerance. Their antifungal spectrum is narrower than posaconazole and probably similar to voriconazole (isavuconazole and ravuconazole) and caspofungin (aminocandin). Licensure and determination of their place in clinical practice requires randomized clinical studies, which are or will be underway.

Polymorphisms in toll-like receptor genes and susceptibility to pulmonary aspergillosis.

Toll-like receptors (TLRs) are important components of innate immunity. We investigated the association between polymorphisms in the TLR2, TLR4, and TLR9 genes and susceptibility to noninvasive forms of pulmonary aspergillosis. A significant association was observed between allele G on Asp299Gly (TLR4) and chronic cavitary pulmonary aspergillosis (odds ratio [OR], 3.46; P =.003). Susceptibility to allergic bronchopulmonary aspergillosis was associated with allele C on T-1237C (TLR9) (OR, 2.49; P =. 043). No particular polymorphism was associated with severe asthma with fungal sensitization. These findings reinforce the importance of innate immunity in the pathogenesis of different forms of aspergillosis.