How to: identify non-tuberculous Mycobacterium species using MALDI-TOF mass spectrometry.

BACKGROUND: The implementation of MALDI-TOF MS for microorganism identification has changed the routine of the microbiology laboratories as we knew it. Most microorganisms can now be reliably identified within minutes using this inexpensive, user-friendly methodology. However, its application in the identification of mycobacteria isolates has been hampered by the structure of their cell wall. Improvements in the sample processing method and in the available database have proved key factors for the rapid and reliable identification of non-tuberculous mycobacteria isolates using MALDI-TOF MS. AIMS: The main objective is to provide information about the proceedings for the identification of non-tuberculous isolates using MALDI-TOF MS and to review different sample processing methods, available databases, and the interpretation of the results. SOURCES: Results from relevant studies on the use of the available MALDI-TOF MS instruments, the implementation of innovative sample processing methods, or the implementation of improved databases are discussed. CONTENT: Insight about the methodology required for reliable identification of non-tuberculous mycobacteria and its implementation in the microbiology laboratory routine is provided. IMPLICATIONS: Microbiology laboratories where MALDI-TOF MS is available can benefit from its capacity to identify most clinically interesting non-tuberculous mycobacteria in a rapid, reliable, and inexpensive manner.

Impact of updating the MALDI-TOF MS database on the identification of nontuberculous mycobacteria.

Conventional identification of mycobacteria species is slow, laborious and has low discriminatory power. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proved highly effective for identifying conventional bacteria, and it may also be useful for identifying mycobacteria. The aim of this study was to evaluate and compare MALDI-TOF MS with currently recommended molecular methods for the identification of nontuberculous mycobacteria (NTM), applying Mycobacteria Libraries v3.0 (ML3.0) and v2.0 (ML2.0). A total of 240 clinical isolates of 41 NTM species grown on solid media were analysed: 132 isolates of slow-growing mycobacteria and 108 of rapid-growing mycobacteria. MALDI-TOF MS, using ML3.0, identified 192 (80%) NTM isolates with a score ≥1.7, encompassing 35 (85.4%) different species, that is, 17 (7.1%; p = 0.0863) isolates and 15 (36.6%; p = 0.0339) species more than currently recommended molecular techniques (polymerase chain reaction reverse hybridization). All these isolates were correctly identified according to molecular identification methods. The application of ML3.0 also identified 15 (6.2%) NTM isolates more than ML2.0 (p < 0.01). The scores obtained with MALDI-TOF MS using ML3.0 (mean score: 1.960) were higher in 147 (61.2%) isolates than when using ML2.0 (mean score: 1.797; p < 0.01). Three of the species analysed were not included in either database, so they were not recognized by this system. In conclusion, MALDI-TOF MS identified more isolates and species than the recommended polymerase chain reaction reverse hybridization assays. Although the new ML3.0 is not the definitive database, it yielded better results than ML2.0. This shows that the updating of the MALDI-TOF MS database plays an essential role in mycobacterial identification. Copyright © 2017 John Wiley & Sons, Ltd.

Increasing isolation of rapidly growing mycobacteria in a low-incidence setting of environmental mycobacteria, 1994-2015.

To determine trends in incidence and clinical relevance of rapidly growing mycobacteria (RGM) in a low-prevalence region of non-tuberculous mycobacteria. We retrospectively identified all patients with RGM-positive cultures between January 1994 and December 2015. Trends in incidence, clinical significance, and outcomes were assessed. One hundred and forty patients had RGM-positive cultures (116 respiratory and 24 extra-respiratory sources). The incidence of RGM isolates increased steadily from 2003 (0.34 per 100,000) to 2015 (1.73 per 100,000), with an average annual increase of 8.3%. Thirty-two patients (22.9%) had clinical disease, which trended to cluster in the second half of the study period. A positive acid-fast bacilli smear (odds ratio [OR] 97.7, 95 % CI 13.8-689.4), the presence of extra-respiratory isolates (OR 19.4, 95 % CI 5.2-72.7), and female gender (OR 5.9, 95 % CI 1.9-19.1) were independently associated with clinical disease. Cure rates were 73.3 and 87.5% for pulmonary and extra-pulmonary disease respectively. Although the burden of disease remains low, the presence of RGM isolates is increasing in our geographical setting. Whether this rise will be sustained over time and will coincide with an increase in clinical disease, or whether it is merely a cycle in the poorly understood epidemiological behaviour of environmental mycobacteria, will be seen in the near future.

Detection of interleukin-2 is not useful for distinguishing between latent and active tuberculosis in clinical practice: a prospective cohort study.

OBJECTIVE: Previous reports have identified interleukin-2 (IL-2), quantified in the supernatants of QuantiFERON®-TB Gold In-tube (QFT) after 72 h of incubation, as a potential biomarker for distinguishing between latent and active tuberculosis (TB). However, its validity has not been tested in an appropriate clinical cohort. METHODS: A multicentre study of 161 consecutive adult patients undergoing evaluation for active TB at eight TB Units in Spain. Interferon-γ (IFN-γ) and IL-2 were assessed in the supernatant of QFT after 16-24 h and 72 h of incubation. The accuracy of IL-2 for indicating latent TB infection (LTBI) was assessed by receiving operating characteristic curves. . RESULTS: Twenty-eight participants were not infected, 43 had LTBI, 69 had TB, and 21 were not classifiable. Median (interquartile range) IL-2 concentrations after 72 h of incubation were 0.0 pg/mL (0.0-0.0) in uninfected individuals, 261.0 pg/mL (81.0-853.0) in LTBI individuals, 166.5 pg/mL (33.5-551.5) in patients with extrapulmonary TB, 95.0 pg/mL (26.0-283.0) in patients with smear-negative pulmonary TB, and 38.5 pg/mL (7.5-178.0) in patients with smear-positive pulmonary TB (p <0.0001). The area under the curve of the receiving operating characteristic curve (95% CI) of IL-2 after 72 h of incubation for the diagnosis of LTBI was 0.63 (0.53-0.74) when all TB cases were considered as a single group, ranging from 0.59 (0.47-0.71) to 0.72 (0.58-0.85) when only extrapulmonary and smear-positive pulmonary TB cases respectively were considered. CONCLUSIONS: Quantification of IL-2 in the supernatant of QFT after a prolonged incubation is not useful to distinguish between LTBI and active disease in clinical practice.

Safe Reduction in CD4 Cell Count Monitoring in Stable, Virally Suppressed Patients With HIV Infection or HIV/Hepatitis C Virus Coinfection.

BACKGROUND: It has been suggested that routine CD4 cell count monitoring in human immunodeficiency virus (HIV)-monoinfected patients with suppressed viral loads and CD4 cell counts >300 cell/µL could be reduced to annual. HIV/hepatitis C virus (HCV) coinfection is frequent, but evidence supporting similar reductions in CD4 cell count monitoring is lacking for this population. We determined whether CD4 cell count monitoring could be reduced in monoinfected and coinfected patients by estimating the probability of maintaining CD4 cell counts ≥200 cells/µL during continuous HIV suppression. METHODS: The PISCIS Cohort study included data from 14 539 patients aged ≥16 years from 10 hospitals in Catalonia and 2 in the Balearic Islands (Spain) since January 1998. All patients who had at least one period of 6 months of continuous HIV suppression were included in this analysis. Cumulative probabilities with 95% confidence intervals were calculated using the Kaplan-Meier estimator stratified by the initial CD4 cell count at the period of continuous suppression initiation. RESULTS: A total of 8695 patients were included. CD4 cell counts fell to <200 cells/µL in 7.4% patients, and the proportion was lower in patients with an initial count >350 cells/µL (1.8%) and higher in those with an initial count of 200-249 cells/µL (23.1%). CD4 cell counts fell to <200 cells/µL in 5.7% of monoinfected and 11.1% of coinfected patients. Of monoinfected patients with an initial CD4 cell count of 300-349 cells/µL, 95.6% maintained counts ≥200 cells/µL. In the coinfected group with the same initial count, this rate was lower, but 97.6% of coinfected patients with initial counts >350 cells/µL maintained counts ≥200 cells/µL. CONCLUSIONS: From our data, it can be inferred that CD4 cell count monitoring can be safely performed annually in HIV-monoinfected patients with CD4 cell counts >300 cells/µL and HIV/HCV-coinfected patients with counts >350 cells/µL.

Polyclonality among clinical strains of non-pigmented rapidly growing mycobacteria: phenotypic and genotypic differences and their potential implications.

To investigate the potential implications (especially the implications in clinical significance and antimicrobial susceptibility) of polyclonality among rapidly growing mycobacteria, we performed random amplified polymorphic DNA analysis in 64 clinical isolates of which the clinical significance was established. Phenotypic characteristics (antimicrobial susceptibility test, colony morphology and growth rate) of each clone were studied. Polyclonality was detected in 13 of the isolates (20.3%). There was a relationship between monoclonality and clinical significance (p 0.0096). Monoclonal and polyclonal isolates showed different behaviour in antimicrobial susceptibility. There was a strong relationship between monoclonality and those species that are more pathogenic for humans, and also with clinical significance of the isolates.

Effect on sperm quality of different cryoprotectants in sperm of Chinchilla lanigera.

Chinchilla lanigera is an endangered species therefore the development of cryopreservation protocols for its gametes is a useful tool in the application of assisted reproduction techniques. A study of the functionality of the spermatozoa punctured from the cauda epididymis was performed on fresh or frozen-thawed samples with three cryoprotective media (test-yolk buffer, sucrose and glycerol). The effect that these media had on sperm physiology during the freezing, storage and later thawing process was analysed. A decrease in the percentages of viability, motility, membrane integrity and capacity to undergo the induced acrosome reaction was found with all the media assayed, an increase in the percentages of DNA fragmentation was also observed. The comparative analysis of the effect of the different cryoprotectants assayed showed that the best medium to use to cryopreserve epididymal sperm in this species is test-yolk buffer. This medium had the least effect on the abovementioned physiological parameters, especially at the level of genetic material.

Evolution of cutaneous tuberculosis over the past 30 years in a tertiary hospital on the European Mediterranean coast.

BACKGROUND: There have been few studies on cutaneous tuberculosis (TB) in Europe in recent years. Objective. To retrospectively analyse the evolution of the various types of cutaneous TB over the past 30 years in an adult population in Spain. METHODS: Patients with cutaneous TB diagnosed between 1981 and 2011 at Bellvitge Hospital, Barcelona, Spain, were included in the study. Chest radiography was performed for all patients, and the presence of TB elsewhere in the body was excluded when clinically suspected. RESULTS: In total, 36 patients (15 male, 21 female, mean age 53.72 years) were diagnosed with cutaneous TB. There were 22 patients with lupus vulgaris (LV), 4 with scrofuloderma, 4 with miliary TB, 3 with tuberculous abscess/ulcer, and 1 each with orificial TB, warty TB, and an iatrogenic inoculation from underlying visceral focus. Of the 36 patients, 16 (38.88%) had TB presenting simultaneously in other organs. Mycobacterial culture from skin biopsies was positive for Mycobacterium tuberculosis complex in 17 of the 32 cases tested (53.12%), whereas stains for acid-fast bacilli in skin samples were positive in only 3 of 36 patients (8.33%). CONCLUSIONS: Although the number of cases of cutaneous TB diagnosed yearly in our population has declined over the past 30 years, cutaneous TB still exists in Europe, and its incidence is expected to increase, owing to the increased immigration into the continent in recent years. The most common type of cutaneous TB in our adult population was LV. It should be noted that despite being considered a benign form of TB, cutaneous TB can be accompanied by TB in internal organs, and severe complications can occur, such as the development of squamous cell carcinoma in long-lasting lesions.

Evaluation of the VersaTREK system compared to the Bactec MGIT 960 system for first-line drug susceptibility testing of Mycobacterium tuberculosis.

The aim of this study was to evaluate the reliability of the VersaTREK system for Mycobacterium tuberculosis drug susceptibility testing compared with results obtained with the Bactec MGIT 960 system. A total of 67 strains were evaluated. Overall agreement was at 98.5%. Kappa indexes were 1.0 for isoniazid, rifampin, and ethambutol, 0.937 for pyrazinamide, and 0.907 for streptomycin. The VersaTREK system is validated for M. tuberculosis drug susceptibility testing.

Tuberculosis transmission patterns among Spanish-born and foreign-born populations in the city of Barcelona.

During a 2-year period (2003-2004), tuberculosis (TB) transmission in Barcelona and the factors related to transmission among the Spanish- and foreign-born populations were studied by molecular epidemiology. Data were obtained from TB cases and Conventional Contact Tracing registries and genotyping was performed using restriction fragment length polymorphism (RFLP)-IS6110 and MIRU12 as a secondary typing method. Of the 892 TB cases reported, 583 (65.3%) corresponded to Spanish-born and 309 (34.6%) to foreign-born. Six hundred and eighty-seven cases (77%) were confirmed by culture. RFLP typing of 463/687 (67.4%) isolates was performed, revealing 280 (60.5%) unique and 183 (39.5%) shared patterns, which were grouped into 65 clusters. Spanish-born individuals were significantly more clustered than foreign-born individuals (44.6% vs. 28.8%; p 0.016). Clustering in foreign-born individuals was associated with HIV (p 0.051, odds ratio = 3.1, 95% confidence interval 1-10.9) and alcohol abuse (p 0.022), whereas, in the Spanish-born individuals, clustering was associated with age in the range 21-50 years, (p 0.024). Of the total clusters, 36/65 (55.3%) included only Spanish-born patients, whereas 22/65 (33.8%) included individuals from both populations. In mixed clusters, the index case was Spanish-born in 53% and foreign-born in 47%. Among the foreign-born, 2.8% were ill on arrival, 30% developed TB within the first year and 50.3% developed TB within the first 2 years; 58.3% were from South America. In conclusion, half of the foreign-born TB patients developed the disease during the first 2 years after arrival, which, in most cases, was the result of endogenous reactivation. Recent TB transmission among Spanish-born and foreign-born populations, as well as bidirectional transmission between communities, contributed significantly to the burden of TB in Barcelona, suggesting the need to improve Public Health interventions in both populations.

Long-term relapses after 12-month treatment for Mycobacterium kansasii lung disease.

Long-lasting therapy for Mycobacterium kansasii lung disease with rifampin-containing multidrug regimens is needed to avoid relapses. The aim of the present study is to evaluate a short multidrug treatment regimen for M. kansasii lung disease. A retrospective observational study of 75 patients with M. kansasii lung disease was conducted in a teaching hospital from January 1990 to December 2005. In total, 75 (67.6%) out of 111 patients diagnosed with M. kansasii lung disease completed a 12-month multidrug treatment regimen, including rifampin, isoniazid and ethambutol, supplemented with streptomycin during the first 2-3 months. After a 41.5-month median follow-up, five (6.6%) patients relapsed. The relapse rate was 2.19 (95% confidence interval 0.71-5.12) per 100 person.yrs. Treatment compliance was considered to be appropriate in all five patients and no drug resistance developed in any case. In conclusion, a 12-month fixed-course treatment is effective in most cases of Mycobacterium kansasii lung disease, but may not be long enough for all patients.

Use of a mycobacteriophage-based assay for rapid assessment of susceptibilities of Mycobacterium tuberculosis isolates to isoniazid and influence of resistance level on assay performance.

We standardized and assessed the performance of an in-house microtiter assay for determining the susceptibilities of Mycobacterium tuberculosis clinical isolates to isoniazid based on mycobacteriophage amplification technology. Seventy isolates (43 resistant and 27 sensitive according to the BACTEC 460 radiometric method and MIC determination) were studied. The isoniazid resistance molecular mechanism was previously determined by sequencing the entire katG gene and the mabA-inhA regulatory region. The sensitivity of the mycobacteriophage-based assay in detecting isoniazid resistance was 86.1%, the specificity achieved was 92.6%, and the overall accuracy was 88.6%. In order to assess the possible influence of resistance levels on the mycobacteriophage-based-assay sensitivity, the results were analyzed according to the isoniazid MICs. All the isolates exhibiting high-level resistance (MIC > or = 2 microg/ml) were scored as resistant by the mycobacteriophage-based assay (100% concordance), and 95% showed mutations or deletions in the catalytic domain of the katG gene. In contrast, 26.1% of the low-level-resistance strains (MICs, 0.25 to 1 microg/ml) were misclassified, and 66.7% had alterations in the mabA-inhA regulatory region. The mycobacteriophage-based assay could be used as a rapid method to detect the isoniazid susceptibility pattern, although data from those areas with high rates of low-level-resistance strains should be interpreted with caution. The features of the assay make it suitable for widespread application due to its low technical demand and cost.

Incidence and molecular typing of Mycobacterium kansasii in a defined geographical area in Catalonia, Spain.

A retrospective population-based study was conducted between January 1990 and December 1998 to investigate the incidence of Mycobacterium kansasji disease and the heterogeneity of the isolates in a well-defined geographical area in Catalonia, Spain. A total of 136 patients were identified. Overall incidence and incidence in AIDS patients was 1.5 (95% CI 1.2-1.8) and 1089.6 (95% CI 689-1330) cases/100,000 persons per year respectively, which is comparable to that reported from most of other geographical areas. Surprisingly, although 7 subtypes of M. kansasii have been consistently reported, in the present study 91 of the 93 isolates (97.8%) tested for genotype were subtype I, regardless of HIV status of the patients. In conclusion, the high rate of infection observed in the AIDS population contributes significantly to the burden of the M. kansasii disease in our area. M. kansasii disease in our geographical area was almost exclusively caused by subtype I regardless of HIV status.

Group B streptococcal disease in nonpregnant adults: incidence, clinical characteristics, and outcome.

A retrospective review of 150 cases of group B streptococcal disease in nonpregnant adults over an 8-year period was performed in a single tertiary-care teaching hospital to determine the incidence, clinical spectrum, and outcome of the disease. Incidence increased from 0.53 cases per 1,000 admissions in the 1993-1994 period to 0.96 cases per 1,000 admissions in 1999-2000 (P=0.013, chi-square test for trend). Bacteremia also increased from 0.15 to 0.42 cases per 1,000 admissions over the same period of time (P=0.005, chi-square test for trend). The mean age of the patients was 61.4 years, and 92% had at least one underlying disease. Bacteremia was detected in 60.9% of patients in whom blood cultures were performed. Fourteen (9.3%) patients died. Factors independently associated with an increased risk of dying were shock at diagnosis (OR, 23.96; 95%CI, 3.44-166.57; P=0.001) and cancer (OR, 4.96; 95%CI, 1.43-17.20; P=0.012). Group B streptococcal disease in nonpregnant adults is on the rise in the hospital investigated, particularly in persons with underlying conditions. The clinical spectrum of the disease ranges from localized to severe bacteremic infections. Shock at diagnosis and cancer are factors independently associated with a higher fatality rate.

Mycobacterium kansasii disease among patients infected with human immunodeficiency virus type 1: improved prognosis in the era of highly active antiretroviral therapy.

OBJECTIVE: To investigate changes in survival and trends in occurrence of Mycobacterium kansasii disease in HIV-infected patients after implementation of highly active antiretroviral therapy (HAART). METHODS: Retrospective analysis of cases of M. kansasii infection diagnosed at the Hospital Universitari de Bellvitge, Barcelona, Spain, between 1991 and 2002. As HAART was introduced at the end of 1996, the study period was divided into periods A (1991-1996) and B (1997-2002). RESULTS: During the study period 44 cases of active M. kansasii disease were identified. The median number of cases per year was five in period A and two in period B. There were no differences in clinical presentation between the two periods. The estimated 24-month survival rate was 16% for period A and 65% for period B (P = 0.0065). Both HAART (RR 11.2; 95% CI 3.8-33.9; P < 0.001) and non-disseminated disease (RR 2.45; 95% CI 1.1-5.2; P = 0.02) were independently associated with longer survival. CONCLUSIONS: A substantial improvement in the outcome and a fall in new cases of M. kansasii in patients infected with HIV-1 was observed in our institution, mainly as a consequence of the introduction of effective antiretroviral therapy. Antiretroviral treatment should be encouraged in these patients, even in cases of severe immunosuppression.

[Prevalence of obesity in hospitalized internal medicine patients]. / Prevalencia de obesidad en pacientes médicos hospitalizados.

OBJECTIVE: We describe the weight's distribution in a sample of medical patients in hospital. We estimate the global prevalence and the presence between other different clinical variables. MATERIAL AND METHODS: A prevalence cross-sectional study was carried out. We determine weight, stature and several clinical variables in 101 patients admit in the internal medicine department of Juan Ramón Jiménez hospital in Huelva. The patients were admitted from 6th to 7th of june in 2000. The Body Mass Index (BMI) > or = 30 Kg/m2 was used to define the obesity. RESULTS: The prevalence of obesity was 32.2% [0.236-0.416]. In the study we find an association with female (prevalente rate -PR- 3.22), HTA (PR 4.72), dislipemia (RP 4.40) and hyperuricacemia (RP 4.28). CONCLUSIONS: The prevalence of obesity in our patients was between 23.41%, it was greater than others estimations in general people. We find association with women and classic cardiovascular risk factors.

Prevalencia de obesidad en pacientes médicos hospitalizados / Prevalence of obesity in an internal medicine department

Fundamento: Se describe la distribución ponderal de una muestra de pacientes médicos hospitalizados. Se estima la prevalencia global y su presencia entre diferentes variables clínicas. Material y métodos: Se diseñó un estudio descriptivo de prevalencia y se determinó el peso, la talla y diversas variables clínicas de 101 pacientes hospitalizados en el Servicio de Medicina Interna del Hospital Juan Ramón Jiménez de Huelva. Los pacientes fueron ingresados entre los días 6-7 de junio de 2000. La obesidad fue definida por un índice de masa corporal (IMC) 30 Kg/m2. Resultados: La prevalencia de la obesidad global fue de 32,7% [0,236 - 0,416]. En el estudio encontramos asociación estadísticamente significativa con el sexo femenino (Razón de prevalencia -RP- 3,22), HTA (RP 4,72), dislipemia (RP 4,40) e hiperuricemia (RP4,28). Conclusiones: La prevalencia de obesidad de nuestros pacientes oscila entre el 23 y 41%, por encima de las estimaciones sobre población general. Más prevalente entre las mujeres y asociada a los factores de riesgo cardiovasculares clásicos (AU)

La infección por el VIH y el sida en el mundo de internet

En la infección por el VIH aparecen avances en cualquiera de sus campos (epidemiología, fisopatología, diagnóstico, prevención y tratamiento) de forma rápida y constante, de manera que la información que se genera va a una velocidad muy superior a la que pueden absorber los medios de difusión convencionales (revistas biomédicas y libros) y en muchas ocasiones la relevancia de la misma tiene implicaciones en la práctica clínica diaria. Eso hace que el personal médico deba estar permanentemente informado.Internet ha permitido vehiculizar y acercar toda esta información a los profesionales sanitarios. El propósito de este capítulo es orientar al lector especializado en el sida para que pueda encontrar rápidamente lo que quiere en la web. Se indican las direcciones que aportan información clínica y revisan las directrices del tratamiento antirretroviral, y de la profilaxis y tratamiento de las infecciones oportunistas, las que especifican los congresos y simposios sobre el VIH, las de las revistas biomédicas y otras publicaciones y las que permiten obtener información básica, institucional, epidemiológica, de soporte social o de consultas a expertos (AU)

In vitro activities of the new ketolide HMR 3647 (telithromycin) in comparison with those of eight other antibiotics against viridans group Streptococci isolated from blood of neutropenic patients with cancer.

The in vitro activities of the ketolide telithromycin and eight other antibiotics were tested against 77 strains of viridans group streptococci isolated from blood samples of neutropenic patients. Thirty-one (40.3%) of the strains were resistant to penicillin G, and 27 (35.1%) were resistant to erythromycin A. Telithromycin (MIC range of < or =0.03 to 1 microg/ml) was the most active antimicrobial tested. These data suggest that telithromycin could be useful for treatment of viridans group streptococcal bacteremia in neutropenic patients with cancer.

High rates of resistance to cephalosporins among viridans-group streptococci causing bacteraemia in neutropenic cancer patients.

The prevalence of resistance to cephalosporins among viridans-group streptococci causing 88 (18%) cases among 485 bacteraemias in neutropenic cancer patients was studied. Rates of resistance to ceftriaxone, ceftazidime, cefpirome and cefepime were 22, 53, 14 and 34%, respectively. Previous administration of beta-lactam therapy was the only factor significantly associated with bacteraemia due to cephalosporin-resistant strains; only 11 (16%) of 68 patients infected with cephalosporin-susceptible bacteria had received these antibiotics compared with 10 (50%) of 20 patients infected with cephalosporin-resistant bacteria (P = 0.0052).