TREM1 regulates antifungal immune responses in invasive pulmonary aspergillosis.

Pattern recognition receptors (PRRs) are responsible for Aspergillus fumigatus recognition by innate immunity and its subsequent immune signaling. The triggering receptor expressed on myeloid cells 1 (TREM1) is a recently characterized pro-inflammatory receptor constitutively expressed on the surface of neutrophils and macrophages. A soluble form (sTREM1) of this protein that can be detected in human body fluids has been identified. Here we investigated the role of TREM1 during invasive pulmonary aspergillosis (IPA). IPA patients displayed significantly higher levels of sTREM1 in bronchoalveolar lavages when compared to control patients. Functional analysis in TREM1 showed that the levels of sTREM1 and TREM1 pathway-related cytokines were influenced by single nucleotide polymorphisms in TREM1. In addition, we confirmed a role of TREM1 on antifungal host defense against A. fumigatus in a murine model of IPA. TREM1 deficiency increased susceptibility to infection in the immunosuppressed murine host. Deletion of TREM1 showed delayed innate and adaptive immune responses and impaired pro-inflammatory cytokine responses. The absence of TREM1 in primary macrophages attenuated the TLR signaling by altering the expression of both receptor and effector proteins that are critical to the response against A. fumigatus. In this study, and for the first time, we demonstrate the key role for the TREM1 receptor pathway during IPA.

High-Resolution Melting Assay for Genotyping Variants of the CYP2C19 Enzyme and Predicting Voriconazole Effectiveness.

Voriconazole is a triazole antifungal agent recommended as primary treatment for invasive aspergillosis, as well as some other mold infections. However, it presents some pharmacokinetic singularities that lead to a great variability intra- and interindividually, nonlinear pharmacokinetics, and a narrow therapeutic range. Most experts have recommended tracing the levels of voriconazole in patients when receiving treatment. This azole is metabolized through the hepatic enzyme complex cytochrome P450 (CYPP450), with the isoenzyme CYP2C19 being principally involved. Allelic variations (polymorphisms) of the gene that encodes this enzyme are known to contribute to variability in voriconazole exposure. Three different allelic variants, CYP2C19*17, CYP2C19*2, and CYP2C19*3, could explain most of the phenotypes related to the voriconazole metabolism and some of its pharmacokinetic singularities. We designed a rapid molecular method based on high-resolution melting to characterize these polymorphisms in a total of 142 samples, avoiding sequencing. Three PCRs were designed with similar cycling conditions to run simultaneously. The results showed that our method represents a fast, accurate, and inexpensive means to study these variants related to voriconazole metabolism. In clinical practice, this could offer a useful tool to individually optimize therapy and reduce expenses in patients with fungal infections.

Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus.

The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR34, TR46, and TR53 Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.

Resultados del tratamiento médico de la embolia de pulmón en una unidad de corta estancia de un hospital de tercer nivel: ¿es el lugar adecuado? / Results of pulmonary embolism treatment in a tertiary hospital short stay unit. Is this the right place?

Objetivo. Describir la estancia media (EM) de la embolia de pulmón (EP) en una unidad de trombosis (UT) dependiente de una unidad de corta estancia (UCE) de un hospital de tercer nivel. Comparar estos datos con el resto de hospitales de nuestra región, con los del resto de comunidades autónomas (CCAA) y con el mismo hospital durante un año previo a nuestra existencia. Material y método. Estudio observacional retrospectivo descriptivo en el que se incluyeron los pacientes con diagnóstico de EP en el Hospital Clínico Universitario Virgen de la Arrixaca (HCUVA) durante el año 2012. Clasificamos estos datos por servicio hospitalario, calculando la EM. Comparamos esta con la del resto de hospitales de nuestra región, con el resto de CCAA y con nuestros datos en el año 2007, cuando no existía aun la UT. Resultados. Se incluyeron 113 pacientes: 60 (53%) ingresaron en la UT, siendo la EM de 4,39 días, en Oncología, de 7,45, y en Medicina Interna (MI), de 15,38. No hubo ningún fallecido en la UT y solo se produjeron 3 reingresos (5%). Los datos publicados mostraron que la EM en todos los hospitales de nuestra región fue de 8,25 días; en nuestro hospital fue de 5,18 días y en el resto de hospitales, mayor. La CCAA con mejor EM fue el País Vasco con 6,85 días. En el año 2007, hubo 70 pacientes con EP en el HCUVA: 34 (49%) en MI con una EM de 8,50 días, 11 (31%) en Oncología con una EM de 9,64 días y 3 (4,3%) en Neumología, con una EM de 19 días; la mortalidad global fue del 11% y la tasa de reingresos en MI, del 6%. Conclusión. La EM de la EP en la UT en una UCE fue menor que en el resto de servicios de nuestro hospital, menor que en el resto de hospitales de nuestra comunidad, menor que en el resto de CCAA y menor que en cualquier servicio de nuestro hospital en una época anterior a nuestra existencia, sin aumentar la tasa de reingreso ni la mortalidad (AU)

Objectives. To determine the mean stay (MS) of patients with pulmonary embolism (PE) in a thrombosis unit (TU) with a short stay unit (SSU) in a tertiary hospital. To compare the data collected with those of other hospitals in the same region, of other regions (Autonomous Communities [AACC]), and within the same hospital in the year before the SSU opened. Material and methods. A descriptive retrospective observational study that included patients with a diagnosis of PE in the University Hospital Virgen de la Arrixaca (HCUVA) in 2012. These data were classified by hospital department, and used for calculating the mean stay. This was then compared with that of other hospitals in our region, with the rest of the regions, and with the data in 2007 (the last year without a TU). Results. A total of 113 patients with PE were included, 60 (53%) in the TU with an MS of 4.39, in Oncology, 7.45, and Internal Medicine (IM), 15.38 days. There were no deaths in the TU and only 3 (5%) readmissions. Published data showed that the MS in all hospitals in our region was 8.25, 5.18 in our hospital, and higher in the rest of hospitals. The best AACC was the Basque Country with an MS of 6.85 days. In 2007, there were 70 patients with PE in the HCUVA, 34 (49%) in IM, with an MS of 8.50, Oncology 11 (31%) with an MS 9.64, and Chest Diseases 3 (4.3%) with an MS 19 days, and with an overall mortality of 11% and a rate of readmissions in IM of 6%. Conclusion. The mean stay for a PE in the SSU of a TU was lower than in the rest of the hospital departments, lower than the rest hospitals of our region, lower than the rest of the regions, and lower than any department of our hospital before the SSU existed, without increasing the readmission or mortality rate (AU)

[Results of pulmonary embolism treatment in a tertiary hospital short stay unit. Is this the right place?]. / Resultados del tratamiento médico de la embolia de pulmón en una unidad de corta estancia de un hospital de tercer nivel: ¿es el lugar adecuado?

OBJECTIVES: To determine the mean stay (MS) of patients with pulmonary embolism (PE) in a thrombosis unit (TU) with a short stay unit (SSU) in a tertiary hospital. To compare the data collected with those of other hospitals in the same region, of other regions (Autonomous Communities [AACC]), and within the same hospital in the year before the SSU opened. MATERIAL AND METHODS: A descriptive retrospective observational study that included patients with a diagnosis of PE in the University Hospital Virgen de la Arrixaca (HCUVA) in 2012. These data were classified by hospital department, and used for calculating the mean stay. This was then compared with that of other hospitals in our region, with the rest of the regions, and with the data in 2007 (the last year without a TU). RESULTS: A total of 113patients with PE were included, 60 (53%) in the TU with an MS of 4.39, in Oncology, 7.45, and Internal Medicine (IM), 15.38days. There were no deaths in the TU and only 3 (5%) readmissions. Published data showed that the MS in all hospitals in our region was 8.25, 5.18 in our hospital, and higher in the rest of hospitals. The best AACC was the Basque Country with an MS of 6.85days. In 2007, there were 70patients with PE in the HCUVA, 34 (49%) in IM, with an MS of 8.50, Oncology 11 (31%) with an MS 9.64, and Chest Diseases 3 (4.3%) with an MS 19days, and with an overall mortality of 11% and a rate of readmissions in IM of 6%. CONCLUSION: The mean stay for a PE in the SSU of a TU was lower than in the rest of the hospital departments, lower than the rest hospitals of our region, lower than the rest of the regions, and lower than any department of our hospital before the SSU existed, without increasing the readmission or mortality rate.

In vivo efficacy of voriconazole and posaconazole therapy in a novel invertebrate model of Aspergillus fumigatus infection.

The emergence of azole resistance in Aspergillus fumigatus is a clinically important issue in the management of invasive aspergillosis as it could limit therapeutic options. Accurate measurement of in vitro antifungal activity in terms of minimum inhibitory concentration (MIC) is considered of clinical relevance and often gives useful therapeutic information for physicians. However, the lack of in vitro-in vivo correlation is frequent and the observed in vitro phenotype does not always correlate with the in vivo response. In this regard, a wild-type strain and five A. fumigatus cyp51A mutated strains showing different azole susceptibility profiles were used to investigate whether the greater wax moth (Galleria mellonella) is an alternative model to assess the in vivo efficacy of voriconazole and posaconazole. Administration of both azoles improved the survival of larvae infected with susceptible strains. However, those larvae infected with resistant strains did not respond to treatment. The phenotype observed in vitro was found to correlate with the efficacy observed in vivo. Moreover, using this in vivo model, the pharmacodynamic target predicting therapeutic success (AUC(0-24)/MIC) was in the same range as previously described, allowing the use of the G. mellonella model to predict the azole susceptibility profile of A. fumigatus strains.

Clinical validation of a multiplex real-time PCR assay for detection of invasive candidiasis in intensive care unit patients.

BACKGROUND: New techniques, such as those based on multiplex quantitative real-time PCR (MRT-PCR), can improve the detection of invasive candidiasis (IC). METHODS: We prospectively studied 63 intensive care unit patients with suspected IC and 40 healthy controls. Blood cultures and MRT-PCR were performed at day 0 and +2, +7, +14 and +21 days in all patients. In addition, ß-d-glucan (BDG) and Candida albicans germ tube antibody (CAGTA) were quantified. RESULTS: IC was confirmed in 27 patients. Colonization was significantly higher in patients with IC (96% versus 64%, P = 0.002). The sensitivity, specificity, positive predictive value and negative predictive value of MRT-PCR for the diagnosis of IC were 96.3%, 97.3%, 92.8% and 98.7%, respectively. The positive predictive value and specificity were significantly higher for MRT-PCR than for BDG and CATGA. MRT-PCR performed very well, especially in deep-seated IC (sensitivity 90.9% versus 45.4% for blood culture; P = 0.06). As regards the most appropriate clinical sample for DNA amplification, in this study whole blood and serum presented similar results. CONCLUSIONS: MRT-PCR appears to be a useful test for confirming a diagnosis of IC in critically ill patients, especially in those with deep-seated disease. Its high sensitivity and positive predictive value make it a much more efficient tool for the management of IC than other diagnostic procedures and clinical scores.

Performance of panfungal--and specific-PCR-based procedures for etiological diagnosis of invasive fungal diseases on tissue biopsy specimens with proven infection: a 7-year retrospective analysis from a reference laboratory.

A retrospective analysis of real-time PCR (RT-PCR) results for 151 biopsy samples obtained from 132 patients with proven invasive fungal diseases was performed. PCR-based techniques proved to be fast and sensitive and enabled definitive diagnosis in all cases studied, with detection of a total of 28 fungal species.

Candida tropicalis antifungal cross-resistance is related to different azole target (Erg11p) modifications.

Candida tropicalis ranks between third and fourth among Candida species most commonly isolated from clinical specimens. Invasive candidiasis and candidemia are treated with amphotericin B or echinocandins as first-line therapy, with extended-spectrum triazoles as acceptable alternatives. Candida tropicalis is usually susceptible to all antifungal agents, although several azole drug-resistant clinical isolates are being reported. However, C. tropicalis resistant to amphotericin B is uncommon, and only a few strains have reliably demonstrated a high level of resistance to this agent. The resistance mechanisms operating in C. tropicalis strains isolated from clinical samples showing resistance to azole drugs alone or with amphotericin B cross-resistance were elucidated. Antifungal drug resistance was related to mutations of the azole target (Erg11p) with or without alterations of the ergosterol biosynthesis pathway. The antifungal drug resistance shown in vitro correlated very well with the results obtained in vivo using the model host Galleria mellonella. Using this panel of strains, the G. mellonella model system was validated as a simple, nonmammalian minihost model that can be used to study in vitro-in vivo correlation of antifungals in C. tropicalis. The development in C. tropicalis of antifungal drug resistance with different mechanisms during antifungal treatment has potential clinical impact and deserves specific prospective studies.

Efficacy of DNA amplification in tissue biopsy samples to improve the detection of invasive fungal disease.

The performance of a pan-fungal PCR-based technique was evaluated to assess the aetiology of invasive fungal diseases (IFDs). A total of 89 formalin-fixed paraffin-embedded biopsy samples from 84 patients with proven IFD were studied. Culture of tissue was performed in 68 (81%) patients. The sensitivities of the PCR-based technique and microbiological culture of tissues were 89% and 56%, respectively (p <0.01). According to PCR results, Aspergillus species accounted for 67%, Candida species for 13%, zygomycetes for 11%, and rare and emerging fungi for 9%. Aspergillus species were significantly associated with lung samples (79.6%, p <0.01), Mucorales were associated with skin/subcutaneous samples, and Candida species were associated with gastrointestinal samples. Regarding biopsy samples with Aspergillus species, Aspergillus fumigatus DNA was detected in 43 of 50 (86%), and Aspergillus flavus in six of 50 (12%). PCR was positive in 24 of 30 (80%) cases with negative culture. In nine of the 84 patients, the PCR technique failed to amplify the DNA. Six also had negative cultures, and in the remaining three cases culture was positive (Rhizopus microsporus, Rhizopus arrhizus, and Sakseneae vasiformis), suggesting that the PCR technique was not as effective in amplifying the DNA of some species of Zygomycetes. In five cases, there was no correlation between culture results and those obtained with DNA amplification, indicating the possibility of a mixed infection or the presence of colonizer/contaminant microorganisms. In summary, PCR-based techniques for DNA amplification should be implemented in histopathology and microbiology departments, as they appear to be complementary to conventional methods for IFD detection.

Development of a single tube multiplex real-time PCR to detect the most clinically relevant Mucormycetes species.

Mucormycetes infections are very difficult to treat and a delay in diagnosis could be fatal for the outcome of the patient. A molecular diagnostic technique based on Real Time PCR was developed for the simultaneous detection of Rhizopus oryzae, Rhizopus microsporus and the genus Mucor spp. in both culture and clinical samples. The methodology used was Molecular beacon species-specific probes with an internal control. This multiplex real-time PCR (MRT-PCR) was tested in 22 cultured strains and 12 clinical samples from patients suffering from a proven mucormycosis. Results showed 100% specificity and a detection limit of 1 fg of DNA per microlitre of sample. The sensitivity was 100% for clinical cultured strains and for clinical samples containing species detected by the PCR assay. Other mucormycetes species were not detected in clinical samples. This technique can be useful for clinical diagnosis and further studies are warranted.

Incidence of zygomycosis in transplant recipients.

Recently, a remarkable increase in the incidence of zygomycosis has been reported from institutions in the USA and Europe. The use of voriconazole for the treatment of aspergillosis and, less frequently, the use of echinocandins as empirical treatment for invasive fungal infections are thought to be responsible for the increase. In addition, an increased incidence of this infection has been observed in transplant recipients, including both haematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) patients. There are no global surveys on the prevalence or incidence of zygomycosis, but data from individual institutions and countries show that zygomycosis is an emerging infection. The increased incidence of zygomycosis most probably reflects a greater frequency of predisposing factors, such as higher numbers of patients undergoing HSCT and immunosuppressive chemotherapy. In addition, the emergence of rare pathogens as a result of the rise in the use of antifungal therapy against common species can be postulated. Further, the availability of antifungal agents with activity profiles that are more specific for selected fungi increases the necessity of identifying pathogenic fungi; the frequency of Zygomycetes infections may have been underestimated until now because therapeutic decisions did not depend on the precise identification of pathogenic fungi.