Epidemiology of Non-Tuberculous Mycobacteria isolated from clinical specimens in Madrid, Spain, from 2013 to 2017.

The epidemiology of non-tuberculous mycobacteria (NTM) in Spain is largely unknown because systematic reporting is not compulsory. The aim of our study was to describe the frequency and diversity of NTM species in our region and their distribution according to the source sample, gender, and age of the patients. We performed a multicenter study of all NTM isolated in 24 public hospitals in Madrid from 2013 to 2017. A total of 6.923 mycobacteria were isolated: 4535 (65.5%) NTM, and 2.388 (34.5%) Mycobacterium tuberculosis complex (MTB). Overall, 61 different NTM species were identified. The most frequently isolated species were Mycobacterium avium complex (47.7%), M. lentiflavum (12.2%), M. gordonae (9.2%), M. fortuitum (8.9%), and M. abscessus (3.9%). Whereas MTB cases were stable during the study period, the number of NTM isolates increased considerably from 930 isolates in 2013 to 1012 in 2017; a sharp increase occurred in the last year. The rise in NTM isolates was mostly due to M. lentiflavum, M. kansasii, and M. abscessus mainly isolated from respiratory specimens in patients older than 60. The increase in isolation rate of NTM in our region is consistent with the increasing rates reported worldwide in the last decades. The rise in NTM isolates was mainly attributed to M. lentiflavum but it also should be noted the increasing of species with high pathogenic potential such as M. kansasii and M. abscessus.

Epidemiology and factors associated with Extra-pulmonary tuberculosis in a Low-prevalence area.

Background: Tuberculosis is a global public health problem. Extra-pulmonary tuberculosis accounts for an increasing proportion of cases worldwide, although information about epidemiological, clinical, or microbiological factors is lacking. Methods: We conducted a retrospective observational study of tuberculosis cases diagnosed between 2016 and 2021, classified into Pulmonary and Extra-pulmonary tuberculosis. Univariable and multivariable logistic regression models were used to investigate risk factors of Extra-pulmonary tuberculosis. Results: 20.9% of overall cases were classified as Extra-pulmonary tuberculosis, with a rising trend from 22.6% in 2016 to 27.9% in 2021. Lymphatic tuberculosis accounted for 50.6% of cases, followed by pleural tuberculosis (24.1%). 55.4% of cases belonged to foreign-born patients. Microbiological culture tested positive in 92.8% of Extra-pulmonary cases. Logistic regression analysis showed that women were more predisposed to develop Extra-pulmonary tuberculosis (aOR 2.46, 95% CI 1.45-4.20) as well as elderly patients (aged ≥ 65 years) (aOR 2.47, 95% CI 1.19-5.13) and persons with previous history of tuberculosis (4.99, 95% CI 1.40-17.82). Conclusions: Extra-pulmonary Tuberculosis have increased within our study period. A profound decline occurred in 2021 tuberculosis cases, probably due to COVID-19. Women, elderly population, and persons with previous history of tuberculosis are at higher risk of developing Extra-pulmonary tuberculosis in our setting.

Diagnostic performance of Anyplex II MTB/MDR/XDR for detection of resistance to first and second line drugs in Mycobacterium tuberculosis.

PURPOSE: Genotypic methods have considerably improved the diagnosis of multidrug-resistant (MDR) tuberculosis. One of these tests is Anyplex II MTB/MDR/XDR (Anyplex). Our aim was to evaluate the diagnostic performance of this multiplex PCR. METHODS: We conducted our study on 47 MDR tuberculosis and 14 pan-susceptible strains. We evaluated the ability of Anyplex to detect resistance mutations in rpoB (rifampin [RIF]), katG and inhA (isoniazid [INH]), gyrA (fluoroquinolones [FLQ]), and rrs and eis (aminoglycosides [AMG]). We used the agar proportion method as gold standard. We also studied concordance with GenoType MTBDRplus (first line drugs) and MTBDRsl (second line drugs). DNA sequencing was applied to clarify discrepancies. RESULTS: All pan-susceptible strains were susceptible by Anyplex. Sensitivity and specificity of Anyplex for detection of resistance mutations were 97.9% and 100%, respectively, for RIF, 91.5% and 100% for INH, 80% and 100% for FLQ, and 50% and 99.7% for AMG. Concordance with GenoType was perfect for RIF, INH, and FLQ (kappa score, k=1.0) and moderate for AMG (k=0.48). Sensitivity and specificity for detection of MDR tuberculosis were 89.4% and 100%, respectively. DNA sequencing of the phenotypically resistant strains considered as susceptible by Anyplex, confirmed no mutations in the corresponding genes. CONCLUSIONS: Anyplex is a reliable assay for the detection of MDR tuberculosis and shows excellent concordance with GenoType. Anyplex reduces the time to diagnosis of MDR tuberculosis strains, as it is recommended by current guidelines on control of tuberculosis.

Value of matrix-assisted laser desorption ionization-time of flight for routine identification of viridans group streptococci causing bloodstream infections.

Phenotypic tests do not always unequivocally identify some species of viridans group streptococci (VGS). sodA sequence analysis is the most accurate method for identification, although it requires specialized personnel and has not been applied systematically in clinical microbiology laboratory routines. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) is emerging as a rapid alternative for bacterial identification. This study assesses the ability of MALDI-TOF and the API 20 Strep system to identify VGS isolates recovered from blood cultures using sodA sequence analysis as the reference method. All clinically significant VGS isolates recovered from blood cultures between January 2007 and January 2010 were identified by sodA sequence analysis and API 20 Strep. The strains were then tested by MALDI-TOF. Agreement between API 20 Strep/MALDI-TOF and sodA sequence analysis was determined. We examined 124 clinical isolates. Sensitivities of API 20 strep and MALDI-TOF for the species level identification of VGS isolates were, respectively, as follows: 60.5% and 73.4%. Sensitivities of API 20 strep and MALDI-TOF for the group level identification were, respectively, as follows: 70% and 94.3%. The turnaround times to identify VGS isolates by sodA sequence analysis, API 20 Strep and MALDI-TOF were 12-24, 24-48 h and 15 min, respectively. API 20 Strep cannot accurately identify all isolates of VGS. MALDI-TOF appeared to be a rapid and reliable alternative for identification of VGS strains to group level, but was not able to discriminate closely related species of certain groups.

Susceptibility testing to second-line drugs and ethambutol by GenoType MTBDRsl and Bactec MGIT 960 comparing with agar proportion method.

The incidence of multidrug-resistant tuberculosis (MDRTB) is increasing. Rapid detection of resistance to second-line drugs is essential for patient management and efficient control of tuberculosis. The aim of the present study was to assess the ability of the GenoType MTBDRsl DNA strip and the Bactec MGIT 960 assay to detect resistance to second-line drugs and ethambutol in multidrug-resistant clinical isolates using the agar proportion method as a reference technique. Twenty-six Mycobacterium tuberculosis complex isolates identified as multidrug-resistant on the basis of conventional drug susceptibility testing were retrieved from our laboratory archive (1992-2010) for evaluation. The susceptibility of these strains to second-line drugs and ethambutol was tested prospectively using MGIT 960 and GenoType MTBDRsl. The turnaround time for agar proportion, MGIT 960, and GenoType MTBDRsl were, respectively, 21 days, 8 days, and 8 h. Sensitivity values for MGIT 960 and GenoType MTBDRsl were, respectively, ethambutol (85.7, 28.6%), amikacin (50, 75%), and ofloxacin (50, 83.3%). Specificity values were, respectively, ethambutol (73.7, 89.5%), amikacin (72.7, 95.5%), and ofloxacin (100, 100%). Our data show that both methods have significant limitations and cannot replace conventional drug susceptibility testing. The results of resistance testing should be interpreted with caution and confirmed using the reference method.

Prolonged viral shedding in pandemic influenza A(H1N1): clinical significance and viral load analysis in hospitalized patients.

The clinical significance of prolonged viral shedding (PVS) and viral load (VL) dynamics has not been sufficiently assessed in hospitalized patients with pandemic 2009 influenza A(H1N1). We performed a prospective study of adults with confirmed influenza A(H1N1) virus infection admitted to our hospital from 20 September 2009 to 31 December 2009. Consecutive nasopharyngeal swabs were collected every 2 days during the first week after diagnosis, and then every week or until viral detection was negative. Relative VL was measured on the basis of haemagglutinin and RNaseP gene analysis. PVS was defined as positive detection of influenza A(H1N1) virus by real-time RT-PCR at day 7 after diagnosis. We studied 64 patients: 16 (25%) presented PVS. The factors associated with PVS were admission to the intensive-care unit (69% vs. 33%, p 0.02), purulent expectoration (75% vs. 44%, p 0.04), higher dosage of oseltamivir (62.5% vs. 27%, p 0.016), corticosteroid treatment (50% vs. 21%, p 0.05), mechanical ventilation (MV) (50% vs. 12.5%, p 0.004), and longer stay (34 vs. 7 median days, p 0.003). Multivariate analysis revealed the factors independently associated with PVS to be immunosuppression (OR 5.15; 95% CI 1.2-22.2; p 0.03) and the need for MV (OR 11.7; 95% CI 2.5-54.4; p 0.002). VL at diagnosis correlated negatively with age and septic shock. VL dynamics of patients with acute respiratory distress syndrome and/or mortality were very different from those of other patients. PVS was detected in 25% of hospitalized patients with pandemic 2009 influenza A(H1N1) and was strongly associated with immunosuppression and the need for MV. Diagnostic VL and viral clearance varied with the clinical course.