Adequacy and prognostic impact of treatment for severe exacerbation of chronic obstructive pulmonary disease. / Adecuación e impacto pronóstico del tratamiento de las exacerbaciones graves de la enfermedad pulmonar obstructiva crónica.

OBJECTIVE: To define the clinical characteristics of patients hospitalised in pneumology and internal medicine departments for chronic obstructive pulmonary disease (COPD) exacerbation, to assess the compliance with the recommendations of the clinical practice guidelines and to determine the impact on the patients' prognosis. METHODOLOGY: We conducted a retrospective longitudinal study that randomly included patients hospitalised for COPD exacerbation in a tertiary hospital. We collected demographic and clinical variables (degree of dyspnoea and obstruction, previous exacerbations, comorbidities), readmission and mortality data and criteria for compliance with the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines and the Spanish COPD guidelines (GesEPOC). We performed a univariate, multivariate and survival analysis. RESULTS: The study included 108 patients, and the mean age was 71.48±11.65 years. The readmission rate was 26.4% at 3 months and 43.4% at 1 year. The hospital mortality rate was 3.9%, the mortality rate at 3 months was 21.9%, and the mortality rate at 1 year was 27.4%. The patients hospitalised in the internal medicine department had higher mortality during hospitalisation (p=.043), at 3 months (p=.028) and at 1 year (p=.007) compared with the rates for the pneumology department. Overall compliance with the clinical guidelines was 63% for the clinical evaluation (less for the patients in internal medicine: 56.1% vs. 73.8%, p=.063). For the treatment, the compliance was 26.9% for GOLD and 28.7% for GesEPOC. Compliance with the GOLD guidelines in the use of corticosteroids was associated with a lower rate of long-term readmissions (p=.041) and hospital mortality (p=.007) and 3-month mortality (p=.05). CONCLUSIONS: The clinical profile of the patients is currently similar to that previously reported, but their clinical progression was poorer. Overall compliance with the clinical guidelines for drug treatment was low, and only appropriate use of systemic steroids was associated with a reduction in early mortality and in medium-term readmissions.

Anticoagulant treatment of medical patients with complex clinical conditions. / Tratamiento anticoagulante en pacientes médicos con situaciones clínicas complejas.

There is scarce available information on the treatment or prophylaxis with anticoagulant drugs of outpatients with medical diseases and complex clinical conditions. There are no clinical practice guidelines and/or specific recommendations for this patient subgroup, which are frequently treated by internists. Complex clinical conditions are those in which, due to comorbidity, age, vital prognosis or multiple treatment with drugs, a clinical situation arises of disease-disease, disease-drug or drug-drug interactions that is not included within the scenarios that commonly generate the scientific evidence. The objective of this narrative review is collecting and adapting of the clinical guidelines recommendations and systematic reviews to complex clinical conditions, in which the direct application of recommendations based on studies that do not include patients with this complexity and comorbidity could be problematic.

Pneumocystis jirovecii infection in patients with acute interstitial pneumonia. / Infección por Pneumocystis jirovecii en pacientes con neumonía intersticial aguda.

OBJECTIVE: Acute interstitial pneumonia (AIP) is a severe disease of unknown etiology. Pneumocystis jirovecii is an atypical opportunistic fungus able to colonize patients with chronic pulmonary disease and inducing alveolar macrophage activation. The aim of this study was to evaluate the possible association between Pneumocystis jirovecii and AIP. SUBJECTS AND METHODS: The presence of P. jirovecii in bronchoalveolar lavage fluid in the four confirmed cases of AIP identified in a tertiary-care hospital over a period of nine years was studied using a 2-step nested-PCR protocol assay. RESULTS: P. jirovecii was identified in the four cases. None of them had HIV infection. Two of the patients were treated empirically with trimethoprim-sulfamethoxazole, the only survivor was being one of them. CONCLUSIONS: Our data suggest that Pneumocystis could trigger or favor the development of AIP. Further studies are needed to evaluate the role of the pathogen in the physiopathology of this disease.

Delphi-RAND consensus of the Spanish Society of Internal Medicine on the controversies in anticoagulant therapy and prophylaxis in medical diseases. INTROMBIN Project (Uncertainty in thromboprophylaxis in internal medicine). / Consenso Delphi-RAND de la Sociedad Española de Medicina Interna sobre controversias en terapia y profilaxis anticoagulante en enfermedades médicas. Proyecto INTROMBIN (INcertidumbre en TROMBoprofilaxis en Medicina INterna).

The aim of this study was to determine the opinion of internists on the management of anticoagulation and thromboembolism prophylaxis in complex clinical scenarios in which the risk-benefit ratio of surgery is narrow and to develop a consensus document on the use of drugs anticoagulant therapy in this patient group. To this end, we identified by consensus the clinical areas of greatest uncertainty, a survey was created with 20 scenarios laid out in 40 clinical questions, and we reviewed the specific literature. The survey was distributed among the internists of the Spanish Society of Internal Medicine (SEMI) and was completed by 290 of its members. The consensus process was implemented by changing the Delphi-RAND appropriateness method in an anonymous, double-round process that enabled an expert panel to identify the areas of agreement and uncertainty. In our case, we also added the survey results to the panel, a methodological innovation that helps provide additional information on the standard clinical practice. The result of the process is a set of 19 recommendations formulated by SEMI experts, which helps establish guidelines for action on anticoagulant therapy in complex scenarios (high risk or active haemorrhage, short life expectancy, coexistence of antiplatelet therapy or comorbidities such as kidney disease and liver disease), which are not uncommon in standard clinical practice.

Geographical variation in serological responses to recombinant Pneumocystis jirovecii major surface glycoprotein antigens.

The use of recombinant fragments of the major surface glycoprotein (Msg) of Pneumocystis jirovecii has proven useful for studying serological immune responses of blood donors and human immunodeficiency virus (HIV)-positive (HIV(+)) patients. Here, we have used ELISA to measure antibody titres to Msg fragments (MsgA, MsgB, MsgC1, MsgC3, MsgC8 and MsgC9) in sera isolated in the USA (n=200) and Spain (n=326), to determine whether geographical location affects serological responses to these antigens. Blood donors from Seville exhibited a significantly greater antibody titre to MsgC8, and significantly lower responses to MsgC3 and MsgC9, than did Cincinnati (USA) donors. Spanish blood donors (n=162) also exhibited elevated responses to MsgC1, MsgC8 and MsgC9 as compared with Spanish HIV(+) (n=164) patients. HIV(+) patients who had Pneumocystis pneumonia (PcP(+)) exhibited a higher response to MsgC8 than did HIV(+) PcP(-) patients. These data show that geographical location plays a role in responsiveness to Msg fragments. Additionally, these fragments have utility in differentiating HIV(+) PcP and HIV(+) PcP(+) among patient populations.

Prediction and biochemical characterization of intrinsic disorder in the structure of proteolysis-inducing factor/dermcidin.

Proteolysis-inducing factor/dermcidin (PIF/DCD) is a novel human gene, located on chromosome 12, locus 12q13.1, that encodes a secreted 110-amino acid protein. Two transcripts for the protein have been identified in normal skin, breast, placenta and brain, and in various primary and metastatic tumor cells. The putative native-state structure of PIF/DCD has not been resolved. Here, we describe some biochemical features of the soluble recombinant 11-kDa protein produced in Escherichia coli. The native 11-kDa polypeptide displayed an anomalous mobility on 1% SDS-PAGE under reduced conditions and appeared as a single approximately 16-kDa band. Under nonreduced conditions, we detected by mass spectrometry, the presence of multiple peaks corresponding to m/z values of 21 kDa, which we confirmed as a dimeric form with a disulfide bridge between cysteine 34 of each 11-kDa monomer. The native protein exhibited an unusually high susceptibility to proteolytic attack by trypsin, and up to 13 peptides derived from its C-terminus were produced after 5 min of incubation. The secondary structure analysis of PIF/DCD native protein in aqueous solution, by circular dichroism spectroscopy, revealed regions with non-well-defined secondary structure but that acquired alpha-helix and beta-sheet secondary structures in the presence of TFE/water mixtures and micellar and non-micellar SDS molecules. By using PONDR, DisEMBL, DisProt, and GlobPlot computational predictors, we identified a long disorder region at the N-terminus of PIF/DCD amino acid sequence. This segment (from 19-50 residues) is critical for some of its biological activities, including neuron survival. This result is coherent with successive failure of crystallization of the protein. Taken together, these data suggest that the disorder and order transition may be relevant for some biological functions of PIF/DCD.

Dynamic colonisation by different Pneumocystis jirovecii genotypes in cystic fibrosis patients.

Although asymptomatic carriers of Pneumocystis jirovecii with cystic fibrosis (CF) have been described previously, the molecular epidemiology of P. jirovecii in CF patients has not yet been clarified. This study identified the distribution and dynamic evolution of P. jirovecii genotypes based on the mitochondrial large-subunit (mt LSU) rRNA gene. The mt LSU rRNA genotypes of P. jirovecii isolates in 33 respiratory samples from CF patients were investigated using nested PCR and direct sequencing. Three different genotypes were detected: 36.3% genotype 1 (85C/248C); 15.1% genotype 2 (85A/248C); 42.4% genotype 3 (85T/248C); and 6% mixed genotypes. Patients studied during a 1-year follow-up period showed a continuous colonisation/clearance cycle involving P. jirovecii and an accumulative tendency to be colonised with genotype 3.

Prediction and biochemical characterization of intrinsic disorder in the structure of proteolysis-inducing factor/dermcidin

Proteolysis-inducing factor/dermcidin (PIF/DCD) is a novel human gene, located on chromosome 12, locus 12q13.1, that encodes a secreted 110-amino acid protein. Two transcripts for the protein have been identified in normal skin, breast, placenta and brain, and in various primary and metastatic tumor cells. The putative native-state structure of PIF/DCD has not been resolved. Here, we describe some biochemical features of the soluble recombinant 11-kDa protein produced in Escherichia coli. The native 11-kDa polypeptide displayed an anomalous mobility on 1% SDS-PAGE under reduced conditions and appeared as a single ~16-kDa band. Under nonreduced conditions, we detected by mass spectrometry, the presence of multiple peaks corresponding to m/z values of 21 kDa, which we confirmed as a dimeric form with a disulfide bridge between cysteine 34 of each 11-kDa monomer. The native protein exhibited an unusually high susceptibility to proteolytic attack by trypsin, and up to 13 peptides derived from its C-terminus were produced after 5 min of incubation. The secondary structure analysis of PIF/DCD native protein in aqueous solution, by circular dichroism spectroscopy, revealed regions with non-well-defined secondary structure but that acquired á-helix and â-sheet secondary structures in the presence of TFE/water mixtures and micellar and non-micellar SDS molecules. By using PONDR®, DisEMBL™, DisProt, and GlobPlot™ computational predictors, we identified a long disorder region at the N-terminus of PIF/DCD amino acid sequence. This segment (from 19-50 residues) is critical for some of its biological activities, including neuron survival. This result is coherent with successive failure of crystallization of the protein. Taken together, these data suggest that the disorder and order transition may be relevant for some biological functions of PIF/DCD.

Pneumocystis jirovecii colonisation in patients with interstitial lung disease.

A prospective study was conducted to determine the prevalence of colonisation by Pneumocystis jirovecii in 80 consecutive patients who required bronchoscopy and bronchoalveolar lavage (BAL) following suspicion of interstitial lung disease (ILD). The mtLSU rRNA gene of P. jirovecii was identified by nested PCR in BAL samples. Patients with ILDs were divided into three groups: group A comprised those with idiopathic interstitial pneumonias; group B comprised those with sarcoidosis; and group C comprised those with other ILDs. The overall prevalence of P. jirovecii carriage was 33.8%, with colonisation rates of 37.8%, 18.8% and 37% in groups A, B and C, respectively (p not significant). There were more smokers among the carriers, but there were no other significant differences between carriers and non-carriers. The high prevalence of P. jirovecii carriers found among immunocompetent patients with ILDs in Spain suggests a possible role of P. jirovecii in the natural history of these diseases.

Prevalence of colonisation and genotypic characterisation of Pneumocystis jirovecii among cystic fibrosis patients in Spain.

Pneumocystis jirovecii colonisation may occur among cystic fibrosis (CF) patients because of their underlying pulmonary disease. A wide epidemiological analysis was performed among CF patients from Spain to assess the prevalence of P. jirovecii colonisation and the distribution of different genotypes. P. jirovecii was identified by nested PCR targeting the mitochondrial large-subunit rRNA gene from sputum samples or oropharyngeal washes. The genotype was determined by direct sequencing. The prevalence of P. jirovecii colonisation among 88 consecutive CF patients was 21.5%. The polymorphisms identified were 85C/248C (45.4%), 85T/248C (27.2%) and 85A/248C (18.1%); in one case, a mix of genotypes was found. Colonisation was more frequent in subjects aged < 18 years (25.5% vs. 15.1%). Among the patients studied, 20.8% received treatment with azithromycin; all of these patients were colonised with P. jirovecii, but none developed Pneumocystis pneumonia (PcP) during a 1-year follow-up period. Concordance in the colonisation status of siblings suggested a common source of infection or person-to-person transmission.

Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis.

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.

High seroprevalence of Pneumocystis infection in Spanish children.

Pneumocystis infection occurs worldwide, and most individuals test seropositive for Pneumocystis early in childhood. Little is known about the epidemiology of this infection in western Europe. The seroprevalence of Pneumocystis infection in 233 Spanish children was determined in a community study by immunoblot analysis of sera. The overall seroprevalence was 73%, with an age-related increase from 52% at 6 years to 66% at 10 years and 80% at 13 years. The data indicated a high seroprevalence of Pneumocystis infection in healthy Spanish children, thereby demonstrating that this pathogen is widespread in southern Spain.

Crystallization, data collection and phasing of infestin 4, a factor XIIa inhibitor.

Infestin is a protein from Triatoma infestans (kissing bug) composed of seven Kazal-type domains that is further processed to yield several serine protease inhibitors with varying specificities. Infestins 3 and 4 are the last two domains of the infestin gene and are found in vivo in the insect's anterior midgut. The last domain, infestin 4, has been cloned, expressed and purified. Here, the crystallization of infestin 4 using the sitting-drop vapour-diffusion method with PEG 8000 as precipitant is described. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 25.89, b = 45.64, c = 57.41 A. X-ray diffraction data were collected to a maximum resolution of 1.8 A using a synchrotron-radiation source. Initial phases were calculated by molecular replacement using an edited rhodniin molecule as the search model. Structure refinement is in progress.

Climatic factors and Pneumocystis jiroveci infection in southern Spain.

The modes of infection and transmission of Pneumocystis jiroveci remain unclear. This study explored the relationship between the incidence of infection and climatic factors. In total, 536 cases of P. jiroveci infection were identified in the period 1994-1998, with an inverse correlation between the incidence of Pneumocystis pneumonia and the minimum mean ambient temperature (Spearman correlation coefficient: r - 0.30; p 0.02; ARIMA model: r - 0.250, p 0.07). The highest number of cases occurred in winter (anova test, p < 0.05), and there was a clear season-related incidence of P. jiroveci infection.

Pneumocystis jiroveci isolates with dihydropteroate synthase mutations in patients with chronic bronchitis.

Since mutations in the dihydropteroate synthase (DHPS) gene possibly associated with sulfonamide resistance have been reported in patients with Pneumocystis jiroveci (previously carinii) pneumonia, and since P. jiroveci colonization has been recently demonstrated in patients with chronic pulmonary diseases, the present study aimed to investigate the possible occurrence of P. jiroveci DHPS mutations in patients with chronic bronchitis. P. jiroveci colonization was detected in 15 of 37 non-selected patients with chronic bronchitis by amplifying the large subunit of the mitochondrial gene of the ribosomal RNA using nested PCR. DHPS mutations were demonstrated using touchdown PCR and restriction enzyme analysis in two of eight patients with chronic bronchitis and in two of six patients from the same region who had AIDS-associated Pneumocystis pneumonia. In all cases, mutations were observed in subjects with no prior exposure to sulfonamides. These data could have important implications for public health, since (i) P. jiroveci colonization could speed the progression of chronic bronchitis, and (ii) these patients, who are customary sputum producers, could represent a reservoir for sulfonamide-resistant strains with the potential ability to transmit them to immunocompromised hosts susceptible to Pneumocystis pneumonia.

Pneumocystis jiroveci genotypes in the Spanish population.

This study describes the genotype distribution of Pneumocystis jiroveci in 79 respiratory samples obtained from 15 patients with acquired immunodeficiency syndrome (AIDS) with P. jiroveci pneumonia and 64 human immunodeficiency virus-negative subjects with different chronic pulmonary diseases. The genotyping was based in analysis of 2 independent genetic loci: the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) fragment (assessed by direct sequencing) and the gene for dihydropteroate synthase (DHPS; assessed by restriction fragment-length polymorphism). The mt LSU rRNA analysis revealed the presence of 3 different polymorphisms for both populations. The major genotype, 85C/248C, was found to be significantly higher in patients with AIDS and P. jiroveci pneumonia than in patients with pulmonary disease. The rate of genotypes 85A/248C and 85T/248C was similar in both groups. The analysis of DHPS genotypes assesses the prevalence of its 4 possible genotypes, with 35.5% of genotypes related to sulfa resistance. The data suggest a common source of infection between both groups.