Microbiological diagnostic procedures for respiratory cystic fibrosis samples in Spain: towards standard of care practices.

BACKGROUND: The microbiological procedures for cystic fibrosis (CF) samples of 17 participating Spanish centers were examined to verify their compliance with current international and national guidelines and to implement the best standards of care for microbiology practices. A 47-item questionnaire covering different CF microbiology aspects was sent to participant laboratories. Telephone interviews were performed when necessary. Data about samples processing for bacteria, mycobacteria and fungi were collected. RESULTS: Gene sequencing (71%), MALDI-TOF (59%) or both (94%) were available for most laboratories. Susceptibility testing was performed by automated microdilution systems (94%) and manual diffusion methods (59%). However, a low use of selective media for Staphylococcus aureus (59%) and Burkholderia cepacia complex (71%), and of epidemiological typing methods (41%) was reported. CONCLUSIONS: Most Spanish laboratories are in agreement with consensus guidelines for the processing of CF respiratory samples, but need to improve in the use of specific selective media and typing methods for epidemiologic studies.

The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study.

A significant knowledge gap exists concerning the geographical distribution of nontuberculous mycobacteria (NTM) isolation worldwide. To provide a snapshot of NTM species distribution, global partners in the NTM-Network European Trials Group (NET) framework (www.ntm-net.org), a branch of the Tuberculosis Network European Trials Group (TB-NET), provided identification results of the total number of patients in 2008 in whom NTM were isolated from pulmonary samples. From these data, we visualised the relative distribution of the different NTM found per continent and per country. We received species identification data for 20 182 patients, from 62 laboratories in 30 countries across six continents. 91 different NTM species were isolated. Mycobacterium avium complex (MAC) bacteria predominated in most countries, followed by M. gordonae and M. xenopi. Important differences in geographical distribution of MAC species as well as M. xenopi, M. kansasii and rapid-growing mycobacteria were observed. This snapshot demonstrates that the species distribution among NTM isolates from pulmonary specimens in the year 2008 differed by continent and differed by country within these continents. These differences in species distribution may partly determine the frequency and manifestations of pulmonary NTM disease in each geographical location.

Changes in the epidemiology of tuberculosis: the influence of international migration flows.

INTRODUCTION: The epidemiological characteristics of patients with tuberculosis (TB) in European hospitals have changed in recent years. METHODS: A prospective study of patients with culture-proven pulmonary TB admitted to our institution from 1997 to 2008 is shown. RESULTS: We analyzed 661 patients with pulmonary TB. An increase in the incidence of TB was confirmed during the study period (P<.001). The proportion of patients with HIV infection decreased from 26% during 1997-2000 to 12% during 2005-2008. However, the proportions of older (>40 years old) and foreign-born patients increased significantly, from 37% to 59% and from 12% to 35%, respectively. Multivariate analysis confirmed previous antituberculous therapy and immigration as factors associated with resistance to isoniazid and to isoniazid+rifampin. After the year 2000, mortality was independently associated with extrapulmonary TB (OR: 3.1; CI 95%: 1.4-7.2), hepatitis C virus infection (OR: 6.0; CI 95%: 2.2-16.3), and diabetes (OR: 6.4; CI 95%: 2.4-16.8). CONCLUSION: Immigration from countries with high rates of TB infection has replaced HIV infection as the most relevant risk factor associated with TB. The increase in the number of older patients with TB and the presence of specific comorbid conditions, especially chronic liver dysfunction, could explain the more difficult management and increased mortality.

[Current panorama of the teaching of microbiology and parasitology in Spain]. / Panorama actual de la docencia de la especialidad de microbiología y parasitología en España.

The training program of residents in microbiology and parasitology in Spain includes clinical skills, ranging from the diagnostic approach to the patient and adequate sample collection for diagnosis of infectious diseases to antimicrobial therapy and infection control measures. Training also includes new challenges in clinical microbiology that ensure residents' participation in infection control programs of health-care associated infections, training in the resolution of public health problems, and application of new molecular microbiology methods. Specialization in clinical microbiology may be undertaken by graduates in Medicine, Biology, Biochemistry and Chemistry. The training is performed in accredited microbiology laboratories at different hospitals (n = 61) across the country through 4-year residency programs. In the last few years, there has been a major imbalance between the number of intended residents (0.17 per 100,000 inhabitants) and those graduating as specialists in clinical microbiology (0.13 per 100,000 inhabitants), with wide variations across the country. The current tendency in Europe is to strengthen the role of clinical microbiologists as key figures in the diagnosis of infectious diseases and in public health microbiology. Training programs have been hampered by the practice of sending samples for microbiological tests to external, centralized multipurpose laboratories with few clinical microbiologists and without a core curriculum. Essential elements in the training of specialists in clinical microbiology are a close relationship between the laboratory and the clinical center and collaboration with other specialists.

Testicular tumor like by "Corynebacterium thommsenii".

OBJECTIVE: To report the first case described of genitourinary infection by Corynebacterium Thommsenii in a man. METHODS: Descriptive study of a testicular infection by an atypical unknown germ in a patient, which was identified and diagnosed thank to the use of Polymerase chain reaction (PCR). We performed a bibliographic search of similar cases. RESULTS: We only found one case of pleural infection by Corynebacterium Thommsenii in the human being, with no case of genitourinary involvement described. CONCLUSIONS: To date there is only one case described of infection by Corynebacterium Thommsenii in human beings, possibly due to underdiagnosis for the slow grow of this pathogen. Genetic amplification methods by PCR should be demanded more frequently by clinicians because they provide an advance in the microbiologic diagnosis is slow-growing pathogens.

Tuberculosis complicating hepatitis C treatment in HIV-infected patients.

Tuberculosis characteristics and incidence were assessed among patients with concurrent human immunodeficiency virus infection and chronic hepatitis C virus infection who were receiving interferon-based therapy at 3 hospitals in Spain. Four of 570 patients (0.7 cases per 100 person-years; 95% confidence interval, 0.19-1.78 cases per 100 person-years) received a diagnosis of tuberculosis; all of them presented with a decrease in CD4+ cell count before diagnosis, and 3 of them received a delayed diagnosis. After tuberculosis treatment, all patients were cured.

Genetic diversity of Mycobacterium avium isolates recovered from clinical samples and from the environment: molecular characterization for diagnostic purposes.

Isolation of Mycobacterium avium complex (MAC) organisms from clinical samples may occur in patients without clinical disease, making the interpretation of results difficult. The clinical relevance of MAC isolates from different types of clinical samples (n = 47) from 39 patients in different sections of a hospital was assessed by comparison with environmental isolates (n = 17) from the hospital. Various methods for identification and typing (commercial probes, phenotypic characteristics, PCR for detection of IS1245 and IS901, sequencing of the hsp65 gene, and pulsed-field gel electrophoresis) were evaluated. The same strain was found in all the environmental isolates, 21 out of 23 (91.3%) of the isolates cultured from urine samples, and 5 out of 19 (26.3%) isolates from respiratory specimens. This strain did not cause disease in the patients. Testing best characterized the strain as M. avium subsp. hominissuis, with the unusual feature that 81.4% of these isolates lacked the IS1245 element. Contamination of certain clinical samples with an environmental strain was the most likely event; therefore, characterization of the environmental mycobacteria present in health care facilities should be performed to discard false-positive isolations in nonsterile samples, mainly urine samples. Molecular techniques applied in this study demonstrated their usefulness for this purpose.

Drug susceptibility of Spanish Mycobacterium tuberculosis complex isolates from animals.

Mycobacterium bovis and Mycobacterium caprae are zoonotic bacteria that cause tuberculosis with several clinical manifestations. We have evaluated the susceptibility to anti-tuberculosis drugs of a panel of Spanish isolates of animal origin. The analysis of the sequence of the main genes involved in resistance was performed in 41 M. bovis and five M. caprae. The katG, inhA, rpsL, embB and gyrA genes had single nucleotide polymorphisms, not previously described in other organisms of the complex. Thirty-two M. bovis and three M. caprae isolates were tested for susceptibility to isoniazid (INH), rifampin, streptomycin, ethambutol, and ofloxacin using the standard proportion method. The results revealed that the isolates were sensitive to the five drugs. However, interference caused by sodium pyruvate in the INH test was detected: 94.3% grew at 0.2 microg INH/ml and 68.6% grew at 1 microg INH/ml. In the medium without pyruvate, 34.3% of the isolates did not grow whereas growth of the others was poor and slow. Nine M. bovis isolates were also tested by ESP Culture System II test and were sensitive to INH. The susceptibility of M. bovis to INH cannot be reliably determined using the standard proportion method due to the M. bovis growth requirements and the interference of pyruvate with INH.

The changing epidemiology of tuberculosis in a Spanish tertiary hospital (1995-2013).

Important epidemiological changes and improvement of new diagnostic approaches, mainly molecular tools, might have impacted the management and outcome of tuberculosis (TB) in the last years in industrialized countries. In order to describe the epidemiological trends, and changes in clinical, diagnostic, and therapeutic aspects in patients with TB, an observational study was performed in a tertiary hospital in Western Europe (Madrid, Spain).All adult patients (>16 years) with a diagnosis of TB in the period 1995 to 2013 were included in the study.TB was diagnosed in 1284 patients, including 304 (24%) foreign-born and 298 (23.2%) human immunodeficiency virus (HIV)-infected patients. The proportion of foreign-born patients increased significantly, from 7.4% (1995) to 40.3% (2013), P < .001, while the proportion of patients with HIV infection decreased (from 41% to 15%, P < .001). Extrapulmonary locations of TB increased (from 23.9% to 37.1%, P < .001), although the miliary forms were less frequent (from 16% to 5.6%, P < .001). Pulmonary involvement remained constant during the period of study (from 50% to 46%, P = .18). The yield of microbiological diagnostic methods in different clinical specimens has remained very similar. Only molecular techniques have improved the diagnosis in respiratory, urinary, and peritoneal samples. The global cure rate was 64.8% and mortality rate was 9.1% (6.5% directly attributable to TB). Mortality has decreased significantly during the years of study (from 11% to 2%, P < .001).There has been a significant decline in the number of patients with TB. Changes in HIV coinfection and immigration have conditioned other epidemiological and clinical aspects of the disease, including the clinical presentation, treatment response, and mortality. Only the use of molecular tests has provided an improvement in the diagnosis of pulmonary and extrapulmonary TB.

Tuberculous orchiepididymitis during 1978-2003 period: review of 34 cases and role of 16S rRNA amplification.

Genitourinary tuberculosis was diagnosed in 543 patients in Spanish hospital from 1978 through 2003. Of the 371 male 34 (9.2%) had orchiepididymitis. Mean age was 52.7 years and the presenting symptom was scrotal swelling and/or pain. Over 50% of cases involved the right ovotestis. Associated renal tuberculosis and active disease in extraurological organs presented in 64% and 19.2% of cases, respectively. Diagnosis was established by culture of Mycobacterium tuberculosis recovery from urine and/or purulent scrotal exudate. Genomic amplification techniques aided the diagnosis in 8 patients. Treatment was rifampin, isoniazide, and pyrazinamide or ethambutol. Eight patients required combined medical and surgical treatment.

Sputum conversion among patients with pulmonary tuberculosis: are there implications for removal of respiratory isolation?

BACKGROUND: Limited data are available to predict the length of time required for a patient to achieve sputum culture conversion after starting therapy for pulmonary tuberculosis. METHODS: Rates of sputum smear and culture conversion were determined at weeks 2, 4, 8 and 16 after initiating therapy in patients admitted to our Respiratory Isolation Unit from January 1997 to December 2003. RESULTS: For the 184 patients included in the analysis, the mean time from the initiation of appropriate therapy to sputum culture and smear conversion were 34 +/- 26 and 38 +/- 32 days (mean +/- SD) respectively. Only 53% of patients obtained negative sputum cultures within the first 4 weeks of therapy. Multivariate analysis showed that the persistence of positive cultures during the first 4 weeks of therapy was associated with high bacillary counts in sputum smears at diagnosis [OR: 2.86; 95% confidence interval (95% CI): 1.20-6.66], lung cavitations (OR: 4.0; 95% CI: 1.63-9.09) and a prolonged period of symptoms (OR: 3.57; 95% CI: 1.43-3.57). The only factor associated with the persistence of positive cultures after more than 16 weeks of therapy was infection with a multidrug-resistant strain. CONCLUSIONS: High initial sputum bacillary counts and drug resistance result in delayed culture conversion. This should be taken into account when decisions regarding the potential discontinuation of isolation are made. The early identification of drug resistance is important for effective infection control in hospitals.

Molecular epidemiology of multidrug-resistant Mycobacterium bovis isolates with the same spoligotyping profile as isolates from animals.

PCR-based characterization techniques have been adopted in most laboratories for Mycobacterium bovis typing. We report a molecular characterization of human multidrug-resistant M. bovis isolates and three bovine isolates that share the spoligotyping profile. The analysis of the direct repeat region showed that both groups differed in the presence of spacers not included in the current membrane. They were also distinguished by two out of the nine mycobacterial interspersed repetitive unit variable-number tandem repeat loci tested, indicating that the human infection was not acquired from the cattle from which isolates were obtained. These results highlight that a combination of techniques is required for appropriate discrimination, even for those spoligotypes that have a low frequency.

Linezolid for the treatment of multidrug-resistant tuberculosis.

OBJECTIVES: In vitro studies have shown good activity of linezolid against Mycobacterium tuberculosis, including multidrug-resistant strains. However, clinical experience with linezolid in tuberculosis is scarce. METHODS: We report our clinical experience with five consecutive patients with multidrug-resistant tuberculosis infection treated with combination regimens that included linezolid. RESULTS: Two patients had multidrug-resistant Mycobacterium bovis infection, with resistance to 12 antituberculous agents (one of them with HIV co-infection and <50 CD4 cells/mm(3)). The other three patients were infected by multidrug-resistant M. tuberculosis strains, with resistance to all first-line drugs and other second-line drugs. All patients received linezolid in combination with thiacetazone, clofazimine or amoxicillin/clavulanate. Susceptibility tests showed linezolid MIC values < or =0.5 mg/L against all tuberculosis strains tested (standard proportion method, Middlebrook agar 7H10). In all cases, tuberculosis cultures from respiratory samples were sterile after 6 weeks of therapy. Three patients have clinical and microbiological cure of tuberculosis with a combination regimen with linezolid (range: 5-24 months). One patient was lost to follow-up at month 5. The remaining patient has completed 11 months of therapy and is still on treatment. Four patients developed anaemia and needed blood transfusions. In two of these patients, the linezolid daily-dose (600 mg twice a day) was successfully reduced to 50% (300 mg twice a day) to decrease toxicity while maintaining efficacy. Peripheral neuropathy (two patients) and pancreatitis (one patient) were other adverse events observed during linezolid treatment. CONCLUSIONS: In our experience, linezolid has been a valid alternative drug in the management of multidrug-resistant tuberculosis. The prolonged use of linezolid is frequently associated with toxicity, mainly anaemia and peripheral neuropathy, that requires special management.

Pseudotumor testicular por "corynebacterium thommseni" / Testicular pseudotumor by “Corynebacterium Thommsenii”

OBJETIVO: Informamos del primer caso descrito en la literatura de infección genitourinaria por Corynebacterium Thomssenii en el hombre.MÉTODOS: Estudio descriptivo de infección testicular por germen atípico y desconocido en un paciente, siendo identificado y diagnosticado gracias a la Reacción en Cadena de la Polimerasa (PCR), realizamos búsqueda en la bibliografía universal de casos similares. RESULTADOS: Encontramos solo un caso de infección pleural por Corynebacterium Thomssenii en el hombre, no estando descritos ningún caso de afección genitourinaria.CONCLUSIONES: Hasta el momento actual no existen más que un caso descrito de infección por C. Thomssenii en el hombre, posiblemente debido a infradiagnostico, por el lento crecimiento de este patógeno. Los métodos de amplificación genética mediante PCR deben ser más demandados por el clínico ya que aportan un adelanto en el diagnóstico microbiológico en patógenos de lento crecimiento(AU)

OBJECTIVE: To report the first case described of genitourinary infection by Corynebacterium Thommsenii in a man.METHODS: Descriptive study of a testicular infection by an atypical unknown germ in a patient, which was identified and diagnosed thank to the use of Polymerase chain reaction (PCR). We performed a bibliographic search of similar cases.RESULTS: We only found one case of pleural infection by Corynebacterium Thommsenii in the human being, with no case of genitourinary involvement described.CONCLUSIONS: To date there is only one case described of infection by Corynebacterium Thommsenii in human beings, possibly due to underdiagnosis for the slow grow of this pathogen. Genetic amplification methods by PCR should be demanded more frequently by clinicians because they provide an advance in the microbiologic diagnosis is slow-growing pathogens(AU)

Geranylgeraniol regulates negatively caspase-1 autoprocessing: implication in the Th1 response against Mycobacterium tuberculosis.

Caspase-1 is a cysteine protease composed by two 20-kDa and two 10-kDa subunits that processes pro-IL-1beta and pro-IL-18 to their mature forms. This enzyme is present in cells as a latent zymogen that becomes active through a tightly regulated proteolytic cascade. Activation is initiated by the oligomerization of an adaptor molecule, or by the formation of a multiprotein complex named inflammasome. Negative regulation of caspase-1 activation is exerted by proteins that compete with the adaptor molecule or with the inflammasome formation. We previously reported that fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, increases caspase-1 activity in PBMC. This effect was strengthened by Mycobacterium tuberculosis, rending an exacerbated IL-1beta, IL-18, and IFN-gamma production. Mevalonate, the product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is a precursor for both nonsterol isoprenoid and sterol formation. In this study, we studied the involvement of mevalonate derivatives in the regulation of caspase-1 activation. Inhibition of sterol formation by SKF-104976 or haloperidol had no effect on IL-1beta release. However, the isoprenoid geranylgeraniol prevented both caspase-1 activation and the exacerbated IL production induced by fluvastatin. This isoprenoid significantly reduced the release of IL-18 and IFN-gamma by PBMC treated with mycobacteria, even in the absence of fluvastatin. In correlation with the increased caspase-1 activity, fluvastatin stimulated the proforms cleavage, enhancing the formation of active subunit p10. Geranylgeraniol not only prevented this effect, but induced proforms accumulation. Present results suggest that, once the proteolytic cascade is initiated, geranylgeraniol may exert an additional negative regulation on caspase-1 cleavage process.

Changes in the epidemiology of tuberculosis: the influence of international migration flows

Introduction: The epidemiological characteristics of patients with tuberculosis (TB) in European hospital shave changed in recent years. Methods: A prospective study of patients with culture-proven pulmonary TB admitted to our institution from 1997 to 2008 is shown. Results: We analyzed 661 patients with pulmonary TB. An increase in the incidence of TB was confirmed during the study period (P<.001). The proportion of patients with HIV infection decreased from 26% during1997-2000 to 12% during 2005-2008. However, the proportions of older (>40 years old) and foreign-bornpatients increased significantly, from 37% to 59% and from 12% to 35%, respectively. Multivariate analysis confirmed previous antituberculous therapy and immigration as factors associated with resistance toisoniazid and to isoniazid+rifampin. After the year 2000, mortality was independently associated with extrapulmonary TB (OR: 3.1; CI 95%: 1.4-7.2), hepatitis C virus infection (OR: 6.0; CI 95%: 2.2-16.3), and diabetes (OR: 6.4; CI 95%: 2.4-16.8).Conclusion: Immigration from countries with high rates of TB infection has replaced HIV infection as the most relevant risk factor associated with TB. The increase in the number of older patients with TB and the presence of specific comorbid conditions, especially chronic liver dysfunction, could explain the more difficult management and increased mortality (AU)

Introducción: La epidemiología de los pacientes con tuberculosis ha cambiado en los hospitales europeos en los últimos años. Métodos: Presentamos un estudio prospectivo en pacientes hospitalizados en nuestra institución con tuberculosis confirmada por cultivo entre los años 1997 y 2008.Resultados: Se analizaron 661 pacientes con tuberculosis pulmonar. Durante el período de estudio se confirmó un incremento de la incidencia (p < 0,001). El porcentaje de pacientes con infección por VIH disminuyó desde el 26% en los años 1997-2000 al 12% en los años 2005-2008. Sin embargo, la proporción de pacientes mayores de 40 años e inmigrantes se incrementó de forma significativa, desde el 37 al 59% y desde el 12 al 35% respectivamente. El análisis multivariante confirmó el tratamiento antituberculoso previo y la inmigración como factores asociados a resistencia a isoniazida y a isoniazida-rifampicina. Después del año 2000 la mortalidad se asoció de forma independiente con la presencia de tuberculosis extrapulmonar (OR: 3,1; IC 95%: 1,4-7,2), infección por virus de la hepatitis C (OR: 6,0; IC 95%: 2,2-16,3),y diabetes (OR: 6,4; IC 95%: 2,4-16,8) (AU)

Panorama actual de la docencia de la especialidad de microbiología y parasitología en España / Current panorama of the teaching of microbiology and parasitology in Spain

El programa de formación de los residentes en microbiología y parasitología recoge su proyección clínica, desde la orientación diagnóstica del paciente y obtención de muestras adecuadas para el diagnóstico de las enfermedades infecciosas, hasta las medidas de tratamiento y control de infección. Asimismo, aborda los nuevos retos de la microbiología clínica y asegura su participación en programas de control de las infecciones asociadas a instituciones de cuidados de salud, la resolución de problemas en salud pública y la aplicación de los nuevos métodos de microbiología molecular. La docencia de la especialidad se realiza a licenciados en medicina, farmacia, biología, bioquímica y química mediante el sistema de residencia (4 años) en 61 unidades docentes acreditadas, presentes en casi todas las comunidades autónomas (CC.AA.). En los últimos años, se han producido desequilibrios importantes entre la oferta de plazas (0,17 por 100.000 habitantes) y los especialistas que terminan su residencia (0,13 por 100.000 habitantes), con una distribución poco homogénea por CC.AA. La tendencia actual en Europa refuerza la figura del microbiólogo clínico como eje central en el área del diagnóstico de las enfermedades infecciosas y su función en el terreno de la microbiología de salud pública. La derivación de pruebas microbiológicas a laboratorios externalizados polivalentes, con escasa participación de especialistas en microbiología y proyectos no sedimentados de troncalidad dificulta la aplicación de los programas de formación. La cercanía a la clínica, en colaboración con otros especialistas, son actitudes inherentes e irrenunciables en la formación de los especialistas en microbiología y parasitología

The training program of residents in microbiology and parasitology in Spain includes clinical skills, ranging from the diagnostic approach to the patient and adequate sample collection for diagnosis of infectious diseases to antimicrobial therapy and infection control measures. Training also includes new challenges in clinical microbiology that ensure residents' participation in infection control programs of health-care associated infections, training in the resolution of public health problems, and application of new molecular microbiology methods. Specialization in clinical microbiology may be undertaken by graduates in Medicine, Biology, Biochemistry and Chemistry. The training is performed in accredited microbiology laboratories at different hospitals (n = 61) across the country through 4-year residency programs. In the last few years, there has been a major imbalance between the number of intended residents (0.17 per 100,000 inhabitants) and those graduating as specialists in clinical microbiology (0.13 per 100,000 inhabitants), with wide variations across the country. The current tendency in Europe is to strengthen the role of clinical microbiologists as key figures in the diagnosis of infectious diseases and in public health microbiology. Training programs have been hampered by the practice of sending samples for microbiological tests to external, centralized multipurpose laboratories with few clinical microbiologists and without a core curriculum. Essential elements in the training of specialists in clinical microbiology are a close relationship between the laboratory and the clinical center and collaboration with other specialists

Nocardiosis sistémica de evolución fatal en una niña previamente sana / Systemic nocardiasis of fatal outcome in a previously healthy female child

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