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.

Characterization of Mycobacterium tuberculosis Beijing isolates from the Mediterranean area.

BACKGROUND: The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases. RESULTS: Only 1% (N = 26) of the isolates from two population-based studies in Spain corresponded to Beijing strains, most of which were pan-susceptible and from Peruvian and Ecuadorian patients. Restriction fragment length polymorphism typing with the insertion sequence IS6110 identified three small clusters (2-3 cases). Mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-15) offered low discriminatory power, requiring the introduction of five additional loci. A selection of the Beijing isolates identified in the Spanish sample, together with a sample of Beijing strains from Italy, to broaden the analysis context in the Mediterranean area, were assayed in an infection model with THP-1 cells. A wide range of intracellular growth rates was observed with only two isolates showing an increased intracellular replication, in both cases associated with contained production of TNF-alpha. No correlation was observed between virulence and the Beijing phylogenetic group, clustered/orphan status, or resistance. The Beijing strain responsible for extensive spread on Gran Canaria Island was also identified in Madrid, but did not lead to secondary cases and did not show high infectivity in the infection model. CONCLUSIONS: The Beijing lineage in our area is a non-homogeneous family, with only certain highly virulent representatives. The specific characterization of Beijing isolates in different settings could help us to accurately identify the virulent representatives before making general assumptions about this lineage.

Fast update of undetected Mycobacterium chimaera infections to reveal unsuspected cases.

Mycobacterium chimaera is involved in a worldwide alert due to contaminated heater-cooler units. A real-time polymerase chain reaction (RT-PCR)-based procedure was implemented to survey undetected cases of M. chimaera infection. PCR was negative in the 59 prosthetic heart valves from patients with PCR-16SrRNA-negative infective endocarditis. PCR identified M. chimaera in one of 15 clinically significant retrospective Mycobacterium avium-Mycobacterium intracellulare complex isolates, which corresponded to a patient who had undergone heart valve replacement in a different institution. Whole-genome sequencing demonstrated that he was the first case in Spain with involvement of the strain responsible for the global outbreak. These results highlight the relevance of retrospective tracking for undetected M. chimaera infections.

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.

Molecular, epidemiological and infectivity characterisation of a Mycobacterium tuberculosis strain prevalent in Madrid.

The most prevalent strain of Mycobacterium tuberculosis in Madrid, Spain (strain 5) was recovered from 45 cases between 1997 and 2004 and showed a highly homogeneous genetic composition. This strain was not exclusive to Spain, and its spoligotyping signature (ST20) was found in entries from different countries in the SITVIT1 database. Patients infected with strain 5 were more frequently positive for human immunodeficiency virus and autochthonous, and had been in prison more frequently, but strain 5 did not show increased infectivity in an in-vitro model of infection.

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.

A novel strategy based on genomics and specific PCR reveals how a multidrug-resistant Mycobacterium tuberculosis strain became prevalent in Equatorial Guinea 15 years after its emergence.

OBJECTIVE: Molecular epidemiology techniques in tuberculosis (TB) can identify high-risk strains that are actively transmitted. We aimed to implement a novel strategy to optimize the identification and control of multidrug-resistant (MDR) TB in a specific population. METHODS: We developed a strain-specific PCR tailored from whole genome sequencing (WGS) data to track a specific MDR prevalent strain in Equatorial Guinea (EG-MDR). RESULTS: The PCR was applied prospectively on remnants of GeneXpert reaction mixtures owing to the lack of culture facilities in Equatorial Guinea. In 147 (93%) of 158 cases, we were able to differentiate between infection by the EG-MDR strain or by any other strain and found that 44% of all rifampicin-resistant TB cases were infected by EG-MDR. We also analysed 93 isolates obtained from Equatorial Guinea 15 years ago, before MDR-TB had become the problem it is today. We found that two of the scarce historical MDR cases were infected by EG-MDR. WGS revealed low variability-six single nucleotide polymorphisms acquired by this strain over 15 years-likely because of the lack in the country of a specific program to treat MDR-TB. CONCLUSIONS: Our novel strategy, which integrated WGS analysis and strain-specific PCRs, represents a low-cost, rapid and transferable strategy that allowed a prospective efficient survey and fast historical analysis of MDR-TB in a population.

Evaluation of alternatives to RFLP for the analysis of clustered cases of tuberculosis.

BACKGROUND: Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis is a recently developed method which could be suitable as a 'real-time' genotyping tool for Mycobacterium tuberculosis. METHODS: One hundred and thirty-four M. tuberculosis isolates were analysed using the reference method, IS6110-restriction fragment length polymorphism (RFLP), and by MIRU, alone and together with spoligotyping. RESULTS: MIRU reduced the genotyping turnaround time by 21 days. The discriminatory power (HGDI) for MIRU and RFLP was 0.978 and 0.989, respectively. RFLP clustered 41.8% of the isolates (17 clusters; 2-9 representatives), whereas MIRU increased the number and size of the clusters (57.5% of the isolates in 20 clusters; 2-14 representatives). With respect to the RFLP clusters, MIRU data showed full correlation in only 7/ 17 (41%) clusters and low correlation in 8/17 (47%) clusters. When MIRU and spoligotyping were considered together, the analysis fitted better with RFLP data: 1) 42.5% of the isolates were grouped in 20 clusters of 2-6 representatives, and 2) the number of clusters with full correlation with RFLP data increased to 11/17 and those with low correlation decreased to 2/17. CONCLUSION: MIRU-VNTR analysis showed low correlation with RFLP. The addition of spoligotyping to MIRU analysis fitted much better with RFLP analysis, although full correlation was still not achieved.

Tuberculosis trends in Madrid, 1994-2003: impact of immigration and HIV infection.

SETTING: Tuberculosis (TB) cases reported from nine districts of Madrid, where the percentage of immigrant population varied from 1.9% in 1996 to 12.2% in 2003. OBJECTIVE: To describe the trends in TB incidence from 1994 to 2003. DESIGN: Observational study. RESULTS: Between 1994-1995 and 2002-2003, the TB rate decreased from 48.5 (95% CI 45.8-51.1) to 23.3 per 100000 population (95% CI 21.5-25.1) (P < 0.001). The percentage of TB cases co-infected with HIV decreased from 55.9% in 1994 to 14.3% in 2003 (P < 0.001), whereas TB cases in foreigners increased from 2.6% in 1994 to 33.7% in 2003 (P < 0.001). CONCLUSION: Although the TB rates showed a marked decrease in the study period, the increasing impact of immigration contributed to slowing down the trend.

Revealing hidden clonal complexity in Mycobacterium tuberculosis infection by qualitative and quantitative improvement of sampling.

The analysis of microevolution events, its functional relevance and impact on molecular epidemiology strategies, constitutes one of the most challenging aspects of the study of clonal complexity in infection by Mycobacterium tuberculosis. In this study, we retrospectively evaluated whether two improved sampling schemes could provide access to the clonal complexity that is undetected by the current standards (analysis of one isolate from one sputum). We evaluated in 48 patients the analysis by mycobacterial interspersed repetitive unit-variable number tandem repeat of M. tuberculosis isolates cultured from bronchial aspirate (BAS) or bronchoalveolar lavage (BAL) and, in another 16 cases, the analysis of a higher number of isolates from independent sputum samples. Analysis of the isolates from BAS/BAL specimens revealed clonal complexity in a very high proportion of cases (5/48); in most of these cases, complexity was not detected when the isolates from sputum samples were analysed. Systematic analysis of isolates from multiple sputum samples also improved the detection of clonal complexity. We found coexisting clonal variants in two of 16 cases that would have gone undetected in the analysis of the isolate from a single sputum specimen. Our results suggest that analysis of isolates from BAS/BAL specimens is highly efficient for recording the true clonal composition of M. tuberculosis in the lungs. When these samples are not available, we recommend increasing the number of isolates from independent sputum specimens, because they might not harbour the same pool of bacteria. Our data suggest that the degree of clonal complexity in tuberculosis has been underestimated because of the deficiencies inherent in a simplified procedure.

LCx: a diagnostic alternative for the early detection of Mycobacterium tuberculosis complex.

This study aims to evaluate the performance of a new diagnostic method (LCx Tuberculosis Assay, Abbott Laboratories) based on Ligase Chain Reaction (LCR) technology, for the detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens and compare it with standard microbiological data and the clinical diagnosis of tuberculosis. Nine hundred specimens were collected from patients with a high suspicion of tuberculosis (740 respiratory samples and 160 non-respiratory specimens). The study was divided into two separate groups: samples washed and distilled water (207 samples) and unwashed samples that were directly resuspended in phosphate buffer (693 samples). The overall sensitivity, specificity, positive and negative predictive values of samples washed with distilled water after decontamination with SDS-NaOH were: 54%, 100%, 100%, and 94%, respectively. If these results were divided according to origin of specimens, the sensitivity, specificity, positive and negative predictive values in respiratory and non-respiratory samples were 54.5%, 100%, 100%, 94% and 50 100%, 100%, 93%, respectively. In contrast, for the non-washed samples, values were 85%, 95%, 80% and 98%, respectively. Respiratory and non-respiratory samples gave values of 84%, 96%, 77%, and 97.5% versus 89%, 99%, 94%, and 98%. The LCx M. tuberculosis assay is a novel, semi-automated assay and a rapid and highly specific technique for screening all forms of tuberculosis, including non-respiratory forms.

Evaluation of the upgraded amplified Mycobacterium tuberculosis direct test (gen-probe) for direct detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens.

We evaluated the upgraded Amplified Mycobacterium Tuberculosis Direct Test kit (AMTD) (Gen-Probe Inc.) for the direct detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens, and compared the results between the traditional 30,000 RLUs cutoff criteria (C) and three equivocal ranges (30,000-100,000, R1; 30,000-500,000, R2; and 30,000-1,000,000, R3). We tested 663 respiratory and 238 non-respiratory samples from 464 patients. The gold standard was considered to be the combination of culture and clinical data. One hundred and nineteen samples were from 56 patients with pulmonary tuberculosis, and 36 samples were from 19 patients with extrapulmonary tuberculosis. When C criteria was applied, the sensitivity and specificity values were 90.8 and 93.0% for respiratory specimens, while they were 88.9 and 92.1% for non-respiratory specimens (p = NS). The sensitivity was significantly higher in smear-positive specimens (96.7%) than in smear-negative ones (81.0%) (p < 0.05). When compared with C criteria, the overall sensitivity was maintained at 90.3% for R1 criteria, and slightly decreased to 89.7% for R2 and R3 criteria (p = NS). Overall, specificity increased significantly from 92.9% (C) to 97.5% (R1), 99.1% (R2), and 99.2% (R3). Application of R2 or R3 criteria improved significantly the specificity of the test with little decrease in sensitivity.

Evaluation of bone marrow and blood cultures for the recovery of mycobacteria in the diagnosis of disseminated mycobacterial infections.

This study evaluated the validity of bone marrow (BM) and blood specimens for the diagnosis of disseminated mycobacterial infections (DMIs). From 1990 to February 1997, all specimens were processed with the lysis-centrifugation procedure; thereafter (until December 2001), they were processed with the BACTEC Myco/F Lytic system. Twenty-three paired BM-blood specimens with mycobacteria in at least one specimen were studied from 23 patients. The strains isolated were 14 Mycobacterium avium complex (MAC) and nine M. tuberculosis complex (MTBC). Blood specimens had a statistically significant greater sensitivity for the isolation of MAC than BM (100% vs. 57.1%, respectively), whereas sensitivity for the isolation of MTBC was equal for the two specimen types (66.7%). Although not statistically significant, the times required to detect mycobacteria from blood specimens were lower than those from BM in the MycoF/Lytic system. Overall, blood cultures represented a more sensitive and less invasive alternative to BM cultures for the diagnosis of disseminated mycobacteriosis caused by MAC, especially when the MycoF/Lytic system was used, but provided no advantage for the diagnosis of DMI caused by MTBC.

Differential findings regarding molecular epidemiology of tuberculosis between two consecutive periods in the context of steady increase of immigration.

The demographic characteristics of the population of Madrid, with a steady increase in immigrants, from 4.7% in 1998 to 17.4% in 2007, provide an opportunity to study in depth the transmission of TB. Our aim was to compare two 3-year longitudinal molecular studies of TB to define transmission patterns and predictors of clustering. Two prospective population-based molecular and epidemiological studies (2002-2004 and 2005-2007) of TB patients were conducted in nine urban districts in Madrid using the same methodology. During the period 2002-2007, 2248 cases of TB were reported, and the incidence decreased from 23.5 per 100,000 in 2002 to 20.8 in 2007 (p <0.001). A total of 1269 isolates were molecularly characterized and included in the study. The comparison between the two periods showed that the percentage of foreign-born patients among TB cases increased from 36.2% to 45.7% (p <0.001). Furthermore, the percentage of clustered cases decreased (36.6% vs. 30.6%; p 0.028), and this decline was associated with a decrease of clustered cases among men and people under 35 years. We also observed a decrease in cases belonging to clusters containing ≥ 6 people (14.2% vs. 8.2%; p <0.001), and in cases belonging to mixed clusters containing Spanish-born and foreign-born patients (18.5% vs. 11.1%, p <0.001). Our molecular epidemiology study provides clues to interpret the decrease in the incidence of TB in a context of steady increase of immigration. In our region, the decrease in the incidence of TB can be explained predominantly as a result of a decline in recent transmission.

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.

[Diagnosis of tuberculosis in paediatrics]. / Diagnóstico de la tuberculosis en la edad pediátrica.

Tuberculosis is one of the most important health problems worldwide. There are an increasing number of cases, including children, due to different reasons in developed countries. The most likely determining cause is immigration from highly endemic areas. Measures to optimise early and appropriate diagnosis of the different forms of tuberculosis in children are a real priority. Two Societies of the Spanish Paediatric Association (Spanish Society of Paediatric Infectology and Spanish Society of Paediatric Pneumology) have agreed this Consensus Document in order to homogenise diagnostic criteria in paediatric patients.

[Diagnosis of tuberculosis in pediatrics. Consensus document of the Spanish Society of Pediatric Infectology (SEIP) and the Spanish Society of Pediatric Pneumology (SENP)]. / Diagnóstico de la tuberculosis en la edad pediátrica. Documento de consenso de la Sociedad Española de Infectología Pediátrica (SEIP) y la Sociedad Española de Neumología Pediátrica (SENP).

Tuberculosis is one of the most important health problems worldwide. There are an increased number of cases, including children, due to different reasons in developed countries. The most likely determining cause is immigration coming from high endemic areas. Measures to optimize early and appropriate diagnosis of the different forms of tuberculosis in children are a real priority. Two Societies of the Spanish Pediatric Association (Spanish Society of Pediatric Infectology and Spanish Society of Pediatric Pneumology) have agreed this Consensus Document in order to homogenize diagnostic criteria in pediatric patients.

Re-analysis of epidemiologically linked tuberculosis cases not supported by IS6110-RFLP-based genotyping.

Tuberculosis microepidemics are considered as such when a proven epidemiological link is identified between the cases. However, some studies have found microepidemics that were not supported by genotyping data. In a cross-sectional study, 44 linked pairs from 33 microepidemics identified during a 5-year period in Madrid, Spain were analysed to evaluate whether the epidemiological findings were consistent with the molecular analysis by IS6110-RFLP. Twelve pairs (27.3%) were not initially confirmed by molecular typing, and a refined re-analysis was performed to identify the reasons for the discrepancies. The possible causes were as follows: (i) laboratory errors or cross-contamination events, (ii) undetected clonally complex infections, and (iii) lack of refinement in the genotyping analysis that could be clarified by applying second-line fingerprinting tools. One discrepant pair was caused by laboratory error. No discrepant pairs were the result of incorrect assignment of genotypes due to clonally complex infections. The application of spoligotyping, MIRU-15 and RFLP enabled the establishment of matching shared genotypes in four linked pairs initially considered as discrepant; therefore, the percentage of discrepant pairs was reduced from 27.3% to 15.9% (7/44). However, in the remaining seven pairs, second-line fingerprinting identified differences with at least two of the three genotyping tools applied. This finding alerts us to the need to (i) refine as much as possible the molecular analysis to establish more accurate identification of truly discrepant cases, and (ii) broaden the search for epidemiological links to include non-conventional contexts outside the household or work/school settings.

Transmission permeability of tuberculosis involving immigrants, revealed by a multicentre analysis of clusters.

In recent years, the number of cases of tuberculosis (TB) among immigrants in Spain has increased markedly, and led to this analysis of the recent transmission patterns of TB in the immigrant population in Madrid. The countries from which the highest number of immigrant cases have been reported were Ecuador (21%), Romania (16%), Morocco (12%), Peru (11%) and Bolivia (9%). Fifty-one per cent of the cases were from South America. In a multicentre study (2004-2006), IS6110 restriction fragment length polymorphism and spoligotyping were used to genotype the Mycobacterium tuberculosis isolates from 632 immigrant cases from 47 countries. A total of 183 cases (29%) were grouped into 59 clusters, which are markers of potential transmission events. Most of the clusters (81%) included patients living in different healthcare districts, and 54% of the clusters were multinational. When a sample of 478 autochthonous cases was included, 53% of the clusters involving immigrants also included autochthonous cases. This study revealed marked transmission permeability among nationalities and between the immigrant and the autochthonous populations.