A strategy based on Amplified Fragment Length Polymorphism (AFLP) for routine genotyping of nontuberculous mycobacteria at the clinical laboratory.

The increasing worldwide incidence of mycobacteriosis and the need to achieve improved clinical management makes nontuberculous mycobacteria (NTM) genotyping a useful tool. However, because of technical difficulties, medium size microbiology laboratories do not attempt to compare the genetic patterns that each of their isolates present. We have aimed to optimize a genotyping method with a reduced hands-on experimental time and that requires few technical resources. A strategy based on the Amplified Fragment Length Polymorphism (AFLP) methodology was developed using two rare-cutters enzymes (SacI and BglII). One out of seven primers was sequentially used in each amplification reaction that was analyzed by agarose gel electrophoresis. This approach makes it possible the timely genotyping of a moderate number of strains and its characterization without the need of image analysis software. We have genotyped 28 Mycobacterium intracellulare and 4 M. abscessus. Clinical researchers are encouraged to routinely genotype their NTM isolates.

Desertomycin G, a New Antibiotic with Activity against Mycobacterium tuberculosis and Human Breast Tumor Cell Lines Produced by Streptomyces althioticus MSM3, Isolated from the Cantabrian Sea Intertidal Macroalgae Ulva sp.

The isolation and structural elucidation of a structurally new desertomycin, designated as desertomycin G (1), with strong antibiotic activity against several clinically relevant antibiotic resistant pathogens are described herein. This new natural product was obtained from cultures of the marine actinomycete Streptomyces althioticus MSM3, isolated from samples of the intertidal seaweed Ulva sp. collected in the Cantabrian Sea (Northeast Atlantic Ocean). Particularly interesting is its strong antibiotic activity against Mycobacterium tuberculosis clinical isolates, resistant to antibiotics in clinical use. To the best of our knowledge, this is the first report on a member of the desertomycin family displaying such activity. Additionally, desertomycin G shows strong antibiotic activities against other relevant Gram-positive clinical pathogens such as Corynebacterium urealyticum, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Clostridium perfringens. Desertomycin G also displays moderate antibiotic activity against relevant Gram-negative clinical pathogens such as Bacteroides fragilis, Haemophilus influenzae and Neisseria meningitidis. In addition, the compound affects viability of tumor cell lines, such as human breast adenocarcinoma (MCF-7) and colon carcinoma (DLD-1), but not normal mammary fibroblasts.

Clinical and Epidemiological Correlates of Low IFN-Gamma Responses in Mitogen Tube of QuantiFERON Assay in Tuberculosis Infection Screening During the COVID-19 Pandemic: A Population-Based Marker of COVID-19 Mortality?

Background: The clinical and epidemiological implications of abnormal immune responses in COVID-19 for latent tuberculosis infection (LTBI) screening are unclear. Methods: We reviewed QuantiFERON TB Gold Plus (QFT-Plus) results (36,709 patients) from July 2016 until October 2021 in Asturias (Spain). We also studied a cohort of ninety hospitalized patients with suspected/confirmed COVID-19 pneumonia and a group of elderly hospitalized patients with COVID-19 who underwent serial QFT-Plus and immune profiling testing. Results: The indeterminate QFT-Plus results rate went from 1.4% (July 2016 to November 2019) to 4.2% during the COVID-19 pandemic. The evolution of the number of cases with low/very low interferon-gamma (IFN-gamma) response in the mitogen tube paralleled the disease activity and number of deaths during the pandemic waves in our region (from March 2020 to October 2021). The percentages of positive QFT-plus patients did not significantly change before and during the pandemic (13.9% vs. 12.2%). Forty-nine patients from the suspected/confirmed COVID-19 pneumonia cohort (54.4%) had low/very low IFN-gamma response to mitogen, 22 of them (24.4%) had severe and critical pneumonia. None received immunosuppressants prior to testing. Abnormal radiological findings (P = 0.01) but not COVID-19 severity was associated with low mitogen response. Immune profiling showed a reduction of CD8 + T cells and a direct correlation between the number of EMRA CD8 + T-cells and IFN-gamma response to mitogen (P = 0.03). Conclusion: Low IFN-gamma responses in mitogen tube of QFT-Plus often occur in COVID-19 pneumonia, which is associated with a low number of an effector CD8 + T-cell subset and does not seem to affect LTBI screening; however, this abnormality seems to parallel the dynamics of COVID-19 at the population level and its mortality.

Diagnosis of Tuberculous Infection in Immunosuppressed Patients and/or Candidates for Biologics Using a Combination of 2 IGRA Tests: T-SPOT.TB/QuantiFERON TB Gold In-Tube vs. T-SPOT.TB/QuantiFERON TB Gold Plus. / Diagnóstico de la infección tuberculosa en pacientes inmunodeprimidos y/o candidatos a terapias biológicas mediante el uso combinado de dos pruebas IGRA: T-SPOT.TB/QuantiFERON TB Gold In-Tube vs. T-SPOT.TB/QuantiFERON TB Gold Plus.

INTRODUCTION: The diagnosis of latent tuberculous infection (LTI) by IGRA continues to generate debate. Experience in the simultaneous use of 2 IGRA tests is scant. The aim of this study was to compare the results of 2 versions of QuantiFERON-TB Gold (In-Tube/Plus) with those of T-SPOT.TB, and to analyse the effectiveness of a dual strategy (T-SPOT.TB + QTF) for the diagnosis of LTI in an immunosuppressed population. METHODS: We conducted a prospective study (May 2015-June 2017) that included 2,999 immunosuppressed patients and/or candidates for biologics, in whom 2 simultaneous IGRA tests were performed: Group 1 (1535 patients): T-SPOT.TB + QuantiFERON-TB Gold-In-Tube (QTF-GIT); Group 2 (1464 patients): T-SPOT.TB + QuantiFERON-TB Gold Plus (QTF-Plus). RESULTS: The concordance between QTF-GIT and T-SPOT.TB was 83.19% (κ=0.532). The percentage of positive, negative, and indeterminate results were, respectively: 14.33% vs. 17.06%; 82.41% vs. 74.46%; and 3.25% vs. 8.46%. The concordance between QTF-Plus and T-SPOT.TB was 87.56% (κ=0.609). The percentage of positive, negative, and indeterminate results were, respectively: 15.02% vs. 15.36%; 82.92% vs. 79.37%; and 2.04% vs. 5.25%. Discrepancies between T-SPOT.TB and QTF-Plus were 12.43%, suggesting that 103 patients were positive and another 79 were negative due exclusively to 1 of the 2 IGRAs. CONCLUSIONS: Greater concordance was found between QTF-Plus and T-SPOT.TB than between QTF-GIT and T-SPOT.TB. However, we believe that the proportion of discrepancies between T-SPOT.TB and QTF-Plus is sufficiently important from a clinical point of view to justify the simultaneous use of 2 IGRA in this specific patient group.

Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification.

The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Löwenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score ≥ 1.6 was obtained for 100% of isolates in 5 laboratories (68.2-98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification.

Buruli ulcers in a Spanish aid worker after a stay in Peru.

Buruli ulcer (BU) is a chronic and destructive infection of the skin and soft tissues caused by Mycobacterium ulcerans. Recently, population flows have triggered the appearance of several sporadic cases of BU in non-endemic countries. This represents a significant diagnostic challenge for clinicians and microbiologists. We describe the first case of BU imported to Spain. The patient was a Spanish woman who had stayed 5 months in the jungle of Peru.

High rates of tuberculin skin test positivity due to methotrexate therapy: False positive results?

RATIONALE: The tuberculin skin test (TST) and interferon ? release assays (IGRAs) are commonly used for latent tuberculosis infection (LTBI) screening. Unexpectedly high TST positivity rates have been reported in patients with rheumatic diseases, and methotrexate is frequently used in this population. We hypothesized that methotrexate use could be associated with false-positive TST results. OBJECTIVES: To investigate whether treatment with methotrexate and other factors are associated with false-positive TST results in patients with rheumatic diseases. METHODS: Prospective single-center study conducted between April 2013 and March 2016. Adult patients with rheumatic diseases were evaluated with a TST and two IGRAs for LTBI screening. We compared TST and IGRA results in patients treated and not treated with methotrexate and analyzed for factors associated with positive TST results. CONCLUSIONS: Our data suggest false-positive TST results associated with methotrexate therapy. Thus, we recommend against using the TST for LTBI screening in patients receiving methotrexate and the preferential use of IGRAs in such patients. MEASUREMENTS AND MAIN RESULTS: We studied 393 patients with rheumatic diseases, including ankylosing spondylitis (ASP, n = 90), rheumatoid arthritis (RA; n = 120), psoriatic arthritis (PA, n = 126), and other disorders (n = 57). The rate of TST positivity varied across the groups: ASP 22.2%, RA 25%, PA 35.7%, and other disorders (22.8%). Positivity rates were lower with IGRAs. Methotrexate use was associated with a statistically significant two-fold increase in the risk of a positive TST and a dose\x96 response relationship was observed. We found no statistically significant associations between methotrexate use and IGRA test positivity.

Utility of Phenotypic and Genotypic Testing in the Study of Mycobacterium tuberculosis Resistance to First-Line Anti-Tuberculosis drugs. / Utilidad de los métodos fenotípicos y genotípicos en el estudio de resistencias de Mycobacterium tuberculosis a fármacos antituberculosos de primera línea.

OBJECTIVE: To determine the utility of molecular techniques in the diagnosis of resistance and the extent of resistance to first-line drugs in our region. MATERIAL AND METHOD: From 2004 to 2013, 1,889 strains of Mycobacterium tuberculosis complex isolated in Asturias, Spain, were studied using phenotypic (Clinical and Laboratory Standards Institute guidelines) and molecular (INNOLiPA RIF-TB©; GenotypeMDRplus©; GenotypeMDRsl©) sensitivity tests. RESULTS: 1,759 strains (94.52%) were sensitive to all first-line drugs, and 102 strains (5.48%) showed some resistance: 81 strains (4.35%) were resistant to 1 single drug, 14 (0.75%) were polyresistant, and 7 (0.37%) were multiresistant (resistant to rifampicin and isoniazid). In total, 137 resistances were identified: 60 to isoniazid (3.22%), 7 to rifampicin (0.37%), 9 to pyrazinamide (0.48%), 11 to ethambutol (0.59%), and 50 to streptomycin (2.68%). Of the mutations detected, 75.9% (63/83) correlated with resistance, while 24.09% of mutations detected (20/83) were not associated with resistance; 16 of these involved a silent mutation at codon 514 of the rpoB gene. Between 0 and 90% of strains, depending on the drug under consideration, were resistant even when no gene mutations were detected using marketed systems. CONCLUSIONS: Molecular techniques are very useful, particularly for obtaining rapid results, but these must be confirmed with standard phenotypic sensitivity testing. The rate of resistance in our region is low and multi-drug resistantcases (0.37%) are sporadic.

Non-Tuberculous Mycobacteria. An Emerging Threat? / Micobacterias no tuberculosas. ¿Una amenaza emergente?

INTRODUCTION AND OBJECTIVE: Non-tuberculous mycobacteria (NTM) isolates are becoming more common. The main objective of our study was to establish the number and diversity of NTM species in our region and their distribution according to the source sample, age and gender of the patients, and to analyse clinically significant isolates. METHODOLOGY: Prospective study of all NTM isolated in Asturias from 2005 to 2012. Samples were processed following internationally accepted guidelines. Statistical analysis was based on Fisher's exact test for 2×2 contingency tables. RESULTS: A total of 3,284 mycobacteria were isolated: 1,499 Mycobacterium tuberculosis complex (MTB) and 1,785 NTM.During the study, NTM isolation rates increased while MTB isolation decreased. NTM were more frequent in men (P<.001). M.gordonae was the most frequently isolated species but did not cause disease in any case. NTM isolates from 212 patients were associated with clinically significant disease (17.1%). M.kansasii and M.avium were most commonly associated with disease. The number of M.kansasii isolates from men was statistically significant (P<.01). CONCLUSIONS: In our study, NTM isolates increased by 35%, compared with a 21% decline in cases of MTB. Both isolation of NTM and clinically significant cases were more common in men. Only 17.1% of NTM isolates were associated with disease, most commonly M.avium complex and M.kansasii.

Attomolar quantitation of Mycobacterium tuberculosis by asymmetric helicase-dependent isothermal DNA-amplification and electrochemical detection.

A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification to electrochemical detection is described. This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity. As a proof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65°C, providing 3×10(6) amplification after 90 min. Using this system 0.5 aM, corresponding to 15 copies of the target gene in 50 µL of sample, can be successfully detected and reliably quantified under isothermal conditions in less than 4h. The assay has been applied to the detection of M. tuberculosis in sputum, pleural fluid and urine samples. Besides this application, the proposed assays is a powerful and general tool for molecular diagnostic that can be applied to the detection of other specific DNA sequences, taking full advantage of the plethora of genomic information now available.

Outbreak or coincidental cases of tuberculosis? Genotyping provides the clue.

We illustrate how genotyping of mycobacterial strains contributed to the discovery of an undetected outbreak of tuberculosis in a hospital ward, ruling out misleading assumptions of transmission chains. Genotyping should be taken into account in routine tests for the control of tuberculosis.