Mortality and cardiovascular risk in vasculitis ANCA. Importance of hypertension and renal function. Experience from southern Spain.

BACKGROUND: Cardiovascular disease (CVD) is one of the principal causes of death in antineutrophil cytoplasmic antibody-(ANCA)-associated vasculitis (AAV). OBJECTIVES: To evaluate the mortality and it's causes and CVD and its vascular risk factors (VRFs) in AAV patients in Andalusia. METHODS: A multicenter cohort of 220 AAV patients followed-up from 1979 until June 2020 was studied in Andalussia, south of Spain. The information, including socio-demographic and clinical data was recorded retrospectively through chart review. Data was analysed using Chi2, ANOVA and Cox proportional hazards regresion as uni and multivariate test with a 95% confidence interval (CI). RESULTS: During a mean ± standard deviation follow-up of 96.79 ± 75.83 months, 51 patients died and 30 presented at least one CVE. Independent prognostic factors of mortality were age (HR 1.083, p=0.001) and baseline creatinine (HR 4.41, p=0.01). Independent prognostic factors of CVE were age [hazard ratio (HR) 1.042, p=0.005] and the presence of hypertension (HTN) six months after diagnosis (HR 4.641, p=0.01). HTN, diabetes and renal failure, all of these important VRFs, are more prevalent in AAV patients than it is described in matched general population. CONCLUSIONS: Age and baseline renal function, but not CVEs, are predictors of mortality and age and early HTN are independent predictors for having a CVE. CVD screening in AAV patients is demanded.

An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates.

Understanding quorum sensing (QS) and its role in the development of pathogenesis may provide new avenues for diagnosing, surveillance, and treatment of infectious diseases. For this purpose, the availability of reliable and efficient analytical diagnostic tools suitable to specifically detect and quantify these essential QS small molecules and QS regulated virulence factors is crucial. Here, we reported the development and evaluation of antibodies and an enzyme-linked immunosorbent assay (ELISA) for HQNO (2-heptyl-4-quinoline N-oxide), a QS product of the PqsR system, which has been found to act as a major virulence factor that interferes with the growth of other microorganisms. Despite the nonimmunogenic character of HQNO, the antibodies produced showed high avidity and the microplate-based ELISA developed could detect HQNO in the low nM range. Hence, a limit of detection (LOD) of 0.60 ± 0.13 nM had been reached in Müeller Hinton (MH) broth, which was below previously reported levels using sophisticated equipment based on liquid chromatography coupled to mass spectrometry. The HQNO profile of release of different Pseudomonas aeruginosa clinical isolates analyzed using this ELISA showed significant differences depending on whether the clinical isolates belonged to patients with acute or chronic infections. These data point to the possibility of using HQNO as a specific biomarker to diagnose P. aeruginosa infections and for patient surveillance. Considering the role of HQNO in inhibiting the growth of coinfecting bacteria, the present ELISA will allow the investigation of these complex bacterial interactions underlying infections. IMPORTANCE Bacteria use quorum sensing (QS) as a communication mechanism that releases small signaling molecules which allow synchronizing a series of activities involved in the pathogenesis, such as the biosynthesis of virulence factors or the regulation of growth of other bacterial species. HQNO is a metabolite of the Pseudomonas aeruginosa-specific QS signaling molecule PQS (Pseudomonas quinolone signal). In this work, the development of highly specific antibodies and an immunochemical diagnostic technology (ELISA) for the detection and quantification of HQNO was reported. The ELISA allowed profiling of the release of HQNO by clinical bacterial isolates, showing its potential value for diagnosing and surveillance of P. aeruginosa infections. Moreover, the antibodies and the ELISA reported here may contribute to the knowledge of other underlying conditions related to the pathology, such as the role of the interactions with other bacteria of a particular microbiota environment.

Long-term treatment with nebulized colistin in oncological patients with recurrent respiratory infections.

OBJECTIVES: To assess the efficacy of long-term treatment with nebulized colistin in reducing the number of respiratory infections, emergency consultations and hospitalizations in oncological patients. METHODS: A retrospective, observational, single-centre study including patients with solid or haematologic malignancies, or pulmonary GVHD after HSTC who received treatment with nebulized colistin for at least six-months to prevent recurrent respiratory infections (July 2010 to June 2017). RESULTS: Twelve patients were included (median age: 54.4, range: 23-85), 7 with solid malignancies and 5 with haematologic malignancies (2 with pulmonary GVHD). Pseudomonas aeruginosa was the most frequent microorganism in sputum cultures (11/12 patients), all strains were susceptible to colistin. There was a statistically significant reduction (p=0.01) in respiratory infections in the six-month period after starting colistin (median: 1, range: 0-4) compared to the six-month period before (median: 4, range: 1-8). There was also a reduction in emergency consultations (precolistin: median: 1.50, range: 0-3; postcolistin: median: 0, range: 0-3) and hospitalizations (precolistin: median: 1.50, range: 0-3; postcolistin: median: 0, range: 0-3) due to respiratory infections. No colistin-resistant strains were identified. CONCLUSIONS: Long-term treatment with nebulized colistin may be useful to reduce the number of exacerbations in oncological patients with recurrent respiratory infections.

Pertactin-Deficient Bordetella pertussis with Unusual Mechanism of Pertactin Disruption, Spain, 1986-2018.

Bordetella pertussis not expressing pertactin has increased in countries using acellular pertussis vaccines (ACV). The deficiency is mostly caused by pertactin gene disruption by IS481. To assess the effect of the transition from whole-cell vaccine to ACV on the emergence of B. pertussis not expressing pertactin in Spain, we studied 342 isolates collected during 1986-2018. We identified 93 pertactin-deficient isolates. All were detected after introduction of ACV and represented 38% of isolates collected during the ACV period; 58.1% belonged to a genetic cluster of isolates carrying the unusual prn::del(-292, 1340) mutation. Pertactin inactivation by IS481 insertion was identified in 23.7% of pertactin-deficient isolates, arising independently multiple times and in different phylogenetic branches. Our findings support the emergence and dissemination of a cluster of B. pertussis with an infrequent mechanism of pertactin disruption in Spain, probably resulting from introduction of ACV.

[Use of 48-hour ABPM in specialist care. Experience in our centre]. / Empleo de la MAPA de 48 horas en atención especializada. Experiencia en nuestro centro.

Ambulatory blood pressure monitoring (ABPM) is a basic tool in the diagnosis and treatment of hypertension (HT). Validity is based on the number of readings taken and their duration. Our aim was to study in our 48-hour ABPM series whether extending the duration of ABPM to 48 hours is justified. MATERIAL AND METHODS: Retrospective analysis of a case series comprising 81 patients with 48-hour ABPM over a 5-year period (2013 to 2018). We analysed the differences between the first and second day. RESULTS: Eighty-one patients, 44 men, mean age of 52 years (± 18). The mean blood pressure (BP) was higher on the first day (132/77 mmHg vs. 130/76 mmHg, p ≤ .01) and there was also a greater proportion of patients with HT on the first day (59 vs. 50%; p ≤ .05). The patients with chronic kidney disease (CKD) (n = 33) had higher systolic BP (SBP) on the second night (p ≤ .05), a circadian rhythm of higher risk on the second day (dipper 13.6 vs. 86.4%, non-dipper 60.7 vs. 41.7%, and riser 30.3 vs. 18.8%; p ≤ .05), more diabetes (39%, p ≤ .01) and more left ventricular hypertrophy (LVH) (74%, p ≤ .05). CONCLUSIONS: 48-hour ABPM could determine BP readings and circadian rhythm better than 24-hour ABPM, especially in patients with CKD and diabetes, both diseases carrying high cardiovascular risk.

A new protocol reduces median door-to-needle time to the benchmark of 30 minutes in acute stroke treatment.

INTRODUCTION: Recent analyses emphasise that The Benchmark Stroke Door-to-Needle Time (DNT) should be 30min. This study aimed to determine if a new in-hospital IVT protocol is effective in reducing door-to-needle time and correcting previously identified factors associated with delays. MATERIAL AND METHODS: In 2014, we gradually introduced a series of measures aimed to reduce door-to-needle time for patients receiving IVT, and compared it before (2009-2012) and after (2014-2017) the new protocol was introduced. RESULTS: The sample included 239 patients before and 222 after the introduction of the protocol. Median overall door-to-needle time was 27min after the protocol was fully implemented (a 48% reduction on previous door-to-needle time [52min], P<.001)]. Median door-to-needle time was lower when pre-hospital code stroke was activated (22min). We observed a 26-min reduction in the median time from onset to treatment (P<.001). After the protocol was implemented, the "3-hour-effect" did not affect door-to-needle time (P=.98). Computed tomography angiography studies performed before IVT were associated with increased door-to-needle time (P<.001); however, the test was performed after IVT was started in most cases. CONCLUSIONS: Hospital reorganisation and multidisciplinary collaboration brought median door-to-needle time below 30min and corrected previously identified delay factors. Furthermore, overall time from onset to treatment was also reduced and more stroke patients were treated within 90min of symptom onset.

An Immunochemical Approach to Quantify and Assess the Potential Value of the Pseudomonas Quinolone Signal as a Biomarker of Infection.

Quorum sensing (QS) is a bacterial cell density-based communication system using low molecular weight signals called autoinducers (AIs). Identification and quantification of these molecules could provide valuable information related to the stage of colonization or infection as well as the stage of the disease. With this scenario, we report here for the first time the development of antibodies against the PQS (pseudomonas quinolone signal), the main signaling molecule from the pqs QS system of Pseudomonas aeruginosa, and the development of a microplate-based enzyme-linked immunosorbent assay (ELISA) able of quantifying this molecule in complex biological media in the low nanometer range (LOD, 0.36 ± 0.14 nM in culture broth media). Moreover, the PQS ELISA here reported has been found to be robust and reliable, providing accurate results in culture media. The technique allowed us to follow up the PQS profile of the release of bacterial clinical isolates obtained from patients of different disease status. A clear correlation was found between the PQS immunoreactivity equivalents and the chronic or acute infection conditions, which supports the reported differences on virulence and behavior of these bacterial strains due to their adaptation capability to the host environment. The results obtained point to the potential of the PQS as a biomarker of infection and to the value of the antibodies and the technology developed for improving diagnosis and management of P. aeruginosa infections based on the precise identification of the pathogen, appropriate stratification of the patients according to their disease status, and knowledge of the disease progression.

A new pleosporalean fungus isolated from superficial to deep human clinical specimens.

Human infections by pleosporalean fungi (class Dothideomycetes, phylum Ascomycota) are rarely reported. Because their identification is challenging using morphological characterization, several phylogenetic markers must be sequenced for an accurate identification and taxonomical placement of the isolates. Three isolates of clinical origin were phenotypically characterized, but due to the absence of relevant morphological traits, D1-D2 domains of the 28S nrRNA gene (LSU), the internal transcribed spacer region (ITS) of the nrRNA, and fragments of the RNA polymerase II subunit 2 (rpb2) and translation elongation factor 1-alpha (tef1) genes were sequenced to allow a phylogenetic analysis that would solve their phylogenetic placement. That analysis revealed that these isolates did not match any previously known pleosporalean genera, and they are proposed here as the new fungal genus, Gambiomyces. Unfortunately, the isolates remained sterile, which, consequently, made the morphological description of the reproductive structures impossible. Future studies should try to understand the behaviour of this fungus in nature as well as its characteristics as an opportunistic fungal pathogen. Molecular identification is becoming an essential tool for proper identification of Dothideomycetes of clinical origin. LAY ABSTRACT: We describe a new pleosporalen pathogenic fungus, Gambiomyces profunda, found in superficial to deep samples from a human patient. Because all strains remained sterile, the fungus was finally identified following a phylogenetic analysis by using four different molecular markers.

High-Throughput Immunochemical Method to Assess the 2-Heptyl-4-quinolone Quorum Sensing Molecule as a Potential Biomarker of Pseudomonas aeruginosa Infections.

Bacterial quorum sensing (QS) is being contemplated as a promising target for developing innovative diagnostic and therapeutic strategies. Here we report for the first time the development of antibodies against 2-heptyl-4-quinolone (HHQ), a signaling molecule from the pqs QS system of Pseudomonas aeruginosa, involved in the production of important virulent factors and biofilm formation. The antibodies produced were used to develop an immunochemical diagnostic approach to assess the potential of this molecule as a biomarker of P. aeruginosa infection. The ELISA developed is able to reach a detectability in the low nM range (IC50 = 4.59 ± 0.29 nM and LOD = 0.34 ± 0.13 nM), even in complex biological samples such as Müeller Hinton (MH) culture media. The ELISA developed is robust and reproducible and has been found to be specific to HHQ, with little interference from other related alkylquinolones from the pqs QS system. The ELISA has been used to analyze the HHQ production kinetics of P. aeruginosa clinical isolates grown in MH media, pointing to its potential as a biomarker of infection and at the possibility to use the technology developed to obtain additional information about the disease stage.

Consensus Multilocus Sequence Typing Scheme for Pneumocystis jirovecii.

Pneumocystis jirovecii is an opportunistic human pathogenic fungus causing severe pneumonia mainly in immunocompromised hosts. Multilocus sequence typing (MLST) remains the gold standard for genotyping of this unculturable fungus. However, the lack of a consensus scheme impedes a global comparison, large scale population studies and the development of a global MLST database. To overcome this problem this study compared all genetic regions (19 loci) currently used in 31 different published Pneumocystis MLST schemes. The most diverse/commonly used eight loci, ß-TUB, CYB, DHPS, ITS1, ITS1/2, mt26S and SOD, were further assess for their ability to be successfully amplified and sequenced, and for their discriminatory power. The most successful loci were tested to identify genetically related and unrelated cases. A new consensus MLST scheme consisting of four genetically independent loci: ß-TUB, CYB, mt26S and SOD, is herein proposed for standardised P. jirovecii typing, successfully amplifying low and high fungal burden specimens, showing adequate discriminatory power, and correctly identifying suspected related and unrelated isolates. The new consensus MLST scheme, if accepted, will for the first time provide a powerful tool to investigate outbreak settings and undertake global epidemiological studies shedding light on the spread of this important human fungal pathogen.

Isolated cerebral mucormycosis associated with intravenous drug use.

We present an uncommon case of isolated basal ganglia mucormycosis in a patient without any known cause of immunosuppression, but with a history of drug injection. The patient presented a good clinical and radiological response to antifungal treatment without aggressive surgical debridement (liposomal amphotericin B combined with isavuconazole for 4 weeks followed by isavuconazole as maintenance therapy for 10 months).

Detection of Streptococcus pneumoniae antigen in pleural fluid: usefulness of an immunofluorescence-based lateral flow assay for the diagnosis of pneumococcal pneumonia.

The performance of an immunofluorescence-based Streptococcus pneumoniae antigen detection test in pleural fluid (IF-PF) was evaluated. For proven and possible pneumococcal pneumonias global sensitivity and specificity were 92.6 (95 CI 76.6-97.9) and 80 (95 CI 62.7-90.5), respectively, with no significant differences between children and adults. Global diagnostic accuracy of IF-PF was 86% (74.2-93.7), and a substantial k index of concordance with culture/RT-PCR of 0.716 (0.535-0.896). IF-PF might be useful as a rapid complementary test for the etiologic diagnosis of pneumococcal pneumonia.

Timely Diagnosis of Histoplasmosis in Non-endemic Countries: A Laboratory Challenge.

Human histoplasmosis is a fungal infection caused by the inhalation of microconidia of the thermally dimorphic fungi Histoplasma capsulatum. Autochthonous cases of histoplasmosis have been diagnosed in almost every country, but it is considered an endemic infection in specific areas of the world. Many of them are popular travel destinations or the source of migratory movements. Thus, the vast majority of the registered cases in non-endemic countries are imported. They correspond to people having been exposed to the fungus in endemic locations as immigrants, expatriates, transient workers or tourists, with reported cases also associated to organ donation. Misdiagnosis and delays in initiation of treatment are not uncommon in cases of imported histoplasmosis. They are associated to high fatality-rates specially in patients with compromised cellular immunity in which progressive disseminated forms develop. The diagnosis of this infection in non-endemic countries is hampered by the lack of clinical suspicion and a dearth of available diagnostic tools adequate to offer rapid and accurate results. Non-culture-based assays such as nucleic-acid amplification tests present as a suitable alternative in this situation, offering improved sensitivity and specificity, shortened turnaround time, and increased biosafety by avoiding culture manipulation. In non-endemic regions, molecular techniques are being used mainly in laboratories from countries that have registered an increase in the incidence of imported cases. However, the number of published techniques is limited and lack consensus. Efforts are currently under way to standardize nucleic acid amplification-based techniques for its implementation in areas registering a rising number of imported cases.

Taking a look on fungi in cystic fibrosis: More questions than answers.

Cystic fibrosis (CF) is one of the most frequent recessive inherited diseases in western countries. Advances in medical care have led to a substantial increase in the life expectancy of CF patients. Survival beyond adolescence has permitted to see fungi not only as late colonizers, but also as potential pathogens responsible of allergic reactions and chronic infections related to lung function deterioration. The role of fungi, nevertheless, has been overlooked until recently. As a result, a number of questions on their epidemiology, clinical significance, or diagnosis, among others, remain unanswered. Besides more in depth studies about the extent of the deleterious effect of fungi on the CF host, new technologies may provide the key to understand its pathogenic role, its interaction with other microbial components of the respiratory microbiota, and should pave the way to define subsets of patients at risk who would benefit from specific therapy. This review is intended to provide a quick overview on what we know about the presence of fungi in the CF airway and its repercussion in the host, and to point out some of the many knowledge gaps needed to understand and advance in the management of fungi in the airway of CF subjects.

Population dynamics and antigenic drift of Bordetella pertussis following whole cell vaccine replacement, Barcelona, Spain, 1986-2015.

Among the factors associated with the resurgence of whooping cough, special emphasis has been given to pathogen adaptation after the introduction of the acellular vaccine (ACV). To assess the impact of the vaccine transition strategy from whole-cell vaccine (WCV) to ACV on population dynamics of Bordetella pertussis in Barcelona (Spain), we studied 339 isolates collected from 1986 to 2015 by PFGE and multi-locus variable-number tandem repeat analysis (MLVA). Additionally, allelic variants for the pertussis toxin and its promoter, pertactin, type 3 fimbriae and fimbrial serotyping were assessed to determine its antigenic drift. A shift was observed in the B. pertussis population as well as in its antigenic profile concurrently with the introduction of ACV in Barcelona. Four out of the five most prevalent PFGE profiles were replaced by new profiles following the ACV introduction. MLVA type 27 was the dominant genotype, and its frequency increased from 25% to 79.3% after WCV replacement. Antigen typing demonstrated the emergence of prn2, ptxP3, fim3-2 and a shift from the fimbriae 3 to the fimbriae 2 serotypes after the ACV introduction. Our findings support the presence of population and antigenic dynamic changes in B. pertussis likely driven by the introduction of ACV.

Molecular Diagnosis of Pneumocystis jirovecii Pneumonia by Use of Oral Wash Samples in Immunocompromised Patients: Usefulness and Importance of the DNA Target.

Pneumocystis jirovecii pneumonia (PJP) is an important cause of pneumonia in the HIV-negative immunocompromised population, for whom the fungal load is low, the differential diagnosis is difficult, and a bronchoalveolar lavage (BAL) sample is often not readily available. Molecular techniques have improved the microbiological diagnosis in this scenario. The usefulness of two real-time PCR techniques targeting nuclear single-copy and mitochondrial multicopy genes, respectively, applied to oral wash specimens (OW) for PJP diagnosis was assessed, and its accuracy was compared to a BAL fluid-based diagnosis. Immunocompromised patients having PJP in the differential diagnosis of an acute respiratory episode, and from whom OW and BAL or lung biopsy specimens were obtained ≤48 h apart, were retrospectively included. PCRs targeting the dihydropteroate synthase gene (DHPS) and the mitochondrial small-subunit (mtSSU) rRNA gene were performed in paired OW-BAL specimens. Thirty-six patients were included (88.6% HIV negative). Fifteen patients (41.7%) were classified as PJP, and a further 8 were considered P. jirovecii colonized. Quantification of DHPS and mtSSU in BAL fluid showed an accuracy of 96.9% and 93.0%, respectively, for PJP diagnosis, whereas a qualitative approach performed better when applied to OW (accuracy, 91.7%) irrespective of the PCR target studied (kappa = 1). Qualitative molecular diagnosis applied to OW showed an excellent performance for PJP diagnosis regardless of the target studied, being easier to interpret than the quantitative approach needed for BAL fluid.

Phaeohyphomycosis caused by Medicopsis romeroi in solid organ transplant recipients: Report of two cases and comprehensive review of the literature.

Medicopsis romeroi is a melanized coelomycetous fungus, mainly found in tropical and subtropical regions and an uncommon cause of infection in solid organ transplant (SOT) recipients. We describe two cases of SOT recipients diagnosed with phaeohyphomycosis due to M romeroi and provide a comprehensive literature review. These infections should be considered in patients native to tropical countries with a localized skin and soft tissue infection. Sequencing is needed for accurate identification of uncommon melanized fungi. Surgical treatment is recommended to cure the infection and co-adjunctive oral antifungals should be considered.

Update of the role of Nuclear Medicine techniques in the pre-surgical localization of primary hyperparathyroidism. / Actualización sobre el papel de las técnicas de Medicina Nuclear en el diagnóstico de localización prequirúrgico en el hiperparatiroidismo primario.

Primary hyperparathyroidism is one of the most frequent endocrine disorders. Its diagnosis is biochemical. Imaging techniques are not useful for the diagnosis of this pathology; they are just tools for pre-surgical localization. In this continuing education, we will analyze the different imaging modalities used in this indication, focusing on Nuclear Medicine. The most commonly used imaging technique in this context is the parathyroid scintigraphy, nowadays double phase protocol with 99mTc-MIBI and the double tracer with 99mTc-MIBI/99mTc-pertechnetate, associated in the first case to SPECT or SPECT/CT, in an early or late phase. The PET/CT with different tracers is showing good results, especially applied to cases of failure in the pre-surgical scintigraphic localization. We expose the results of the morphological imaging techniques as well as the usefulness of combining techniques.

Immune Parameters for Diagnosis and Treatment Monitoring in Invasive Mold Infection.

Infections caused by invasive molds, including Aspergillus spp., can be difficult to diagnose and remain associated with high morbidity and mortality. Thus, early diagnosis and targeted systemic antifungal treatment remains the most important predictive factor for a successful outcome in immunocompromised individuals with invasive mold infections. Diagnosis remains difficult due to low sensitivities of diagnostic tests including culture and other mycological tests for mold pathogens, particularly in patients on mold-active antifungal prophylaxis. As a result, antifungal treatment is rarely targeted and reliable markers for treatment monitoring and outcome prediction are missing. Thus, there is a need for improved markers to diagnose invasive mold infections, monitor response to treatment, and assist in determining when antifungal therapy should be escalated, switched, or can be stopped. This review focuses on the role of immunologic markers and specifically cytokines in diagnosis and treatment monitoring of invasive mold infections.