Juan J. Palacios

Humans as source of Mycobacterium tuberculosis infection in cattle, Spain.

To the Editor: Mycobacterium tuberculosis is the main causative agent of tuberculosis in humans. However, little attention has been paid to its transmission from humans to animals. We report M. tuberculosis infections in 3 cattle farms in Spain. The epidemiologic investigation traced humans as the source of infection, with 1 of the strains showing multidrug resistance. Recent studies have reported isolation of M. tuberculosis in cattle with prevalences of 4.7%–30.8% in African and Asian countries (1–3). In cattle, this infection occurs in countries with the highest incidence of human tuberculosis in the world. In Europe, only 14 cases of M. tuberculosis infection have been described in 3 eastern countries since implementation of eradication programs (4,5). The only reported cases of M. tuberculosis in cattle in western Europe were described in Great Britain and date back to the 1950s (6). During 2007–2009, three cases of tuberculosis caused by M. tuberculosis were detected in 3 unrelated cattle farms, 2 of them free of tuberculosis (farms 1 and 2). As part of the surveillance system of bovine tuberculosis, a pool of tissue samples from each cow (respiratory lymph nodes and lung) were homogenized with sterile distilled water, and culture was carried out by the BACTEC mycobacteria growth indicator tube 960 system (Beckton Dickinson, Madrid, Spain). Members of the M. tuberculosis complex were identified and genotyped by direct variable repeat spacer olignucleotide typing and mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) typing (7). The 3 M. tuberculosis–infected animals were <9 months of age (Table). As described (6), the possibility of infection in young animals could be more probable than infection in older cows. Table Relevant information about Mycobacterium tuberculosis infection in 3 cattle farms in Spain* M. tuberculosis–infected animals from farms 1 and 3 were detected by the intradermal tuberculin test (Table). The animal without immunologic response (farm 2) was detected because an M. bovis infection was confirmed in the herd, and all animals were slaughtered. Confirmation of infection by culture without immunologic response is rare, although the high sensitivity of the mycobacteria growth indicator tube system could detect a low bacterial load in the initial stages of infection. Recent implementation of liquid systems in animal health laboratories has enabled detection of M. tuberculosis when it is compared with results using only conventional methods. Moreover, no tuberculosis-compatible lesions were observed in the 3 animals, similar to previous studies (6). On the basis of these facts, M. tuberculosis transmission was not detected among cattle in the following intradermal tuberculin tests. Co-infection with other mycobacteria (M. avium subsp. hominissuis) was found in the same animal from farm 1 (Table). This co-infection suggested the immunocompromised status of the animal and hence a high susceptibility to M. tuberculosis infection. Moreover, M. bovis was isolated from 52% (16/31) of all animals from farm 2 that showed a positive reaction to the intradermal tuberculin skin test, making remarkable the absence of co-infection with M. bovis in the M. tuberculosis–infected animal. Therefore, the lack of M. tuberculosis transmission within this herd contrasts with the M. bovis dissemination. The veterinary services reported these findings to the National Public Health System, and an epidemiologic investigation was conducted on the cattle farms to determine the source of infection. In all cases, staff of the farms had active tuberculosis (Table). Three different strains were characterized: SIT2537 (octal code 777617777720771), 253533233433236252211423 (farm 1); SIT1564, 3′52334232455457251213423 (farm 2); and SIT58, 254343243232325262213423 (farm 3) (Table). The MIRU-VNTR pattern and spoligotype are shared by Spanish human and cattle isolates from farm 1; SIT2537 is an uncommon profile that has been detected in Brazil and Spain (according to the SITVIT2 database). The human strain showed multidrug resistance to isoniazid, rifampin, and ethionamide. In cattle and human isolates, genes associated with isoniazid and rifampin resistance were studied (8) and rpoB analysis confirmed rifampin resistance (Ser531Leu). In farm 2, the origin of the farm worker was eastern Europe and the cattle isolate showed an SIT1564 profile, which is found only in 6 human isolates in the SpolDB4 database, all from Poland, Bulgaria, and Russia. On farm 3, human and cattle isolates from Spain shared identical spoligotype and MIRU-VNTR patterns. The profile SIT58 is frequent in Spain (9) and other countries with historical links to Spain, mainly the south American countries (79/114 according to SpolDB4). A well-designed program for eradicating bovine tuberculosis helps to detect M. tuberculosis infection by immune response or by bacteriologic culture. The use of liquid systems and results of epidemiologic studies (Spanish Database of Animal Mycobacteriosis, mycoDB.es) (S. Rodriguez, unpub. data) are recommended for prompt confirmation of the M. tuberculosis complex infection and for enhancing the sensitivity of culture. In addition, the Spanish Ministry of Environment, Rural and Marine Affairs has reinforced the need to improve cooperation between human and animal health systems to minimize the risk for M. tuberculosis complex transmission from animals to humans or vice versa and to control infection in all susceptible animal species (10).

Isolation of Mycobacterium tuberculosis Strains with a Silent Mutation in rpoB Leading to Potential Misassignment of Resistance Category

ABSTRACT Our study provides an alert regarding the transmission of rifampin-susceptible strains of Mycobacterium tuberculosis with a silent substitution in codon 514 of rpoB. Among 1,450 cases, we identified 12 isolates sharing this mutation and related restriction fragment length polymorphism (RFLP) types. The mutation impaired hybridization with the wild-type probes in three independent commercial assays, which could lead to misassignment of resistance.

T-cell profiling and the immunodiagnosis of latent tuberculosis infection in patients with inflammatory bowel disease.

Background:Factors associated with performance of interferon-&ggr; release assays (IGRA) and the tuberculin skin test (TST) in screening for latent tuberculosis infection in patients with inflammatory bowel diseases (IBD) are still poorly understood. The influence of peripheral T-cell subset counts on the results also remain unclear. Methods:Prospective single-center study in 205 patients with IBD. Latent tuberculosis infection screening included a chest radiograph, TST (retest if negative), and 2 IGRAs: QuantiFERON-TB Gold In-Tube (QFT-GIT) and TSPOT-TB (TSPOT). T-cell subpopulations were determined by flow cytometry. Results:Twenty-one (10.2%) patients had an abnormal chest radiograph, 55 (26.8%) had a positive TST, 16 (7.8%) had a positive QFT-GIT, and 25 (12.6%) had a positive TSPOT. TST positivity was lower in patients on ≥2 immunosuppressants compared with the controls (5-aminosalicylic acid treatment) (10.4% versus 38.2%, respectively) (P = 0.0057). No other drugs influenced TST or IGRA positivity. In patients on corticosteroid treatment, anti-TNF treatment, or ≥2 immunosuppressants, IGRAs detected 10 cases of latent tuberculosis infection not identified by TST. TSPOT and QFT-GIT increased yield by 56% and 22%, respectively. No significant differences in T-cell subpopulations were found between patients with positive or negative TST or TSPOT results. However, patients with positive QFT-GIT findings had more CD8+ T cells (mean, 883 ± 576 versus 484 ± 385 cells per microliter in patients with negative results) (P = 0.022). Conclusions:IGRAs can improve TST-based screening in patients with IBD on immunosuppressive therapy. A low CD8+ count can affect QFT-GIT results. We suggest combining TSPOT and TST screening in patients with IBD on immunosuppressants.

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.

Diagnóstico y tratamiento de la tuberculosis con resistencia a fármacos

In the last 2 decades, drug-resistant tuberculosis has become a threat and a challenge to worldwide public health. The diagnosis and treatment of these forms of tuberculosis are much more complex and prognosis clearly worsens as the resistance pattern intensifies. Nevertheless, it is important to remember that with the appropriatesystematic clinical management, most of these patients can be cured. These guidelines itemize the basis for the diagnosis and treatment of all tuberculosis patients, from those infected by strains that are sensitive to all drugs, to those who are extensively drug-resistant. Specific recommendations are given forall cases. The current and future role of new molecular methods for detecting resistance, shorter multi-drug-resistant tuberculosis regimens, and new drugs with activity against Mycobacterium tuberculosis are also addressed.

Strong In Vitro Activities of Two New Rifabutin Analogs against Multidrug-Resistant Mycobacterium tuberculosis

ABSTRACT Two new rifabutin analogs, RFA-1 and RFA-2, show high in vitro antimycobacterial activities against Mycobacterium tuberculosis. MIC values of RFA-1 and RFA-2 were ≤0.02 μg/ml against rifamycin-susceptible strains and 0.5 μg/ml against a wide selection of multidrug-resistant strains, compared to ≥50 μg/ml for rifampin and 10 μg/ml for rifabutin. Molecular dynamic studies indicate that the compounds may exert tighter binding to mutants of RNA polymerase that have adapted to the rifamycins.

Comparison of MB-Check and Löwenstein-Jensen media for recovery of mycobacteria

We have compared the rate of recovery of mycobacteria with the MB‐Check culture system (liquid phase) and the Löwenstein‐Jensen (LJ) medium in 2,907 clinical specimens obtained from 830 patients submitted for mycobacterial culture during 1‐year period. Direct smear examination was carried out by auramine‐rhodamine staining. All primary isolates from the culture media were confirmed by Ziehl‐Neelsen staining and identified by acridinium‐ester‐labeled DNA probes specific for Mycobaterium tuberculosis complex. A total of 214 isolates were of the M. tuberculosis complex (88 patients) and 54 of “potentially pathogenic environmental mycobacteria” (45 patients). A total of 117 (54.7%) samples were smear‐positive and the remaining 97 (45.3%) were smear‐negative. There was a significant difference in the percentage of positive cultures obtained by the MB‐Check method (99.1%) as compared with the LJ medium (73.8%) (P < 0.05). This difference, however, occurred almost exclusively at the expense of the 97 smear‐negative samples (positive cultures 97.95% by the MB‐Check method vs. 42.3% by the LJ culture, P < 0.05). The number of patients diagnosed of tuberculosis by the MB‐Check was significantly higher as compared with LJ medium (88 [100%] vs. 77 [87.5%], P < 0.05). In 11 (12.5%) patients, the diagnosis was only established by the MB‐Check system. In smear‐positive samples, the mean (± SD) detection time for M. tuberculosis complex was 14.8 ± 8 days with MB‐Check and 19.9 ± 7 days with LJ medium. The corresponding figures in smear‐negative samples were 22.8 ± 3 days and 27.8 ± 6 days, respectively. DNA probes directly applied to MB‐Check liquid medium showed a sensitivity of 98.8% and specificity of 100%. These results indicate that the MB‐Check system is more efficient for the recovery of mycobacteria than LJ medium. Microsc. Res. Tech. 38:512–518, 1997.

Molecular and epidemiological population-based integrative analysis of human and animal Mycobacterium bovis infections in a low-prevalence setting

Human Mycobacterium bovis infections are considered to be due to reactivations, when involve elderly people, or to recent transmissions, when exposure is occupational. We determined the cause of M. bovis infections by genotyping M. bovis isolates in a population-based study integrating human and animal databases. Among the 1,586 tuberculosis (TB) cases in Asturias, Northern Spain (1,080,000 inhabitants), 1,567 corresponded to M. tuberculosis and 19 to M. bovis. The number of human isolates sharing genotype with cattle isolates was higher than expected (47%) for a setting with low prevalence of bovine TB and efficient control programs in cattle. The risk of exposure to infected animals was probable/possible in most of these matched cases (77.7%). Recent transmission was the likely explanation of most M. bovis infections in elderly people. A potential human-to-human transmission was found. Our study illustrates a model of collaboration between human and animal health professionals to provide a precise snapshot of the transmission of M. bovis in the human-animal interface.

Comentarios a la normativa SEPAR de diagnóstico y tratamiento de la tuberculosis con resistencia a fármacos

We are grateful to Dr. Pascual for his insightful comments1 on our recently published guidelines on the diagnosis and treatment of drug-resistant tuberculosis (TB).2 His letter addresses 2 points that we would like to discuss further. We fully agree with the first of his comments, regarding the good anti-Mycobacterium tuberculosis activity of bedaquiline and delamanid. As shown in Table 3 of our guidelines,2 the effectiveness of these new drugs prompted us to include them among the core drugs (our group 4) instead of among the add-on agents (Group D) recommended in the 2016 WHO recommendations3; we agree that these 2 drugs should have been included in WHO group C.3 As early as 2015, our group published an article in the European Respiratory Journal, proposing that these new drugs (along with linezolid) should even be positioned above second-line injectable drugs.4 The reason for not including these drugs in treatment regimens from the outset is not so much financial, but because we feel that evidence for such use is still scant. The inclusion of these drugs in group 4 illustrates our view: they are very good but evidence is still lacking. However, by including them in group 4, we are providing the option for their use in MDR-TB regimens. As for shorter MDR-TB regimens, Dr. Pascual rightly points out that many issues remained unclear in the 2016 WHO publication.3 For this reason, and because of concerns surrounding the applicability of these regimens in practice, the WHO subsequently published an article which addressed many of these controversies.5 In it, they stated that the decision to use shorter regimens should not be based on sensitivity testing to the drugs in the regimen, other than fluoroquinolones or second-line injectables. This is because sensitivity results for these drugs, including pyrazinamide, are unreliable. The authors go so far as to say that even if resistance to pyrazinamide is observed, the clinician can consider using these shorter regimens.5 Furthermore, our recommendation not to use these regimens in pregnant women and in extrapulmonary TB is based on a lack of

Evaluation of the potential role of a new mutation in mabA in modifying the response of Mycobacterium tuberculosis to isoniazid

A new mutation in mabA, Thr4Ile, was identified in a Mycobacterium tuberculosis isolate from a patient whose culture remained positive after treatment. The same mutation was found in another 5 patients infected by different strains. A putative role for this mutation in the process of diminishing susceptibility to isoniazid is evaluated.