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.

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.

[Usefulness of chemotherapy associated with surgery in the management of nontuberculous mycobacterial adenitis]. / Utilidad de antibioticoterapia asociada a cirugía en el tratamiento de adenopatías por micobacterias no tuberculosas.

BACKGROUND: In recent years, lymphadenitis caused by atypical mycobacteria (also called nontuberculous mycobacteria [NTMB] or, more recently, environmental) have played a significant role in the differential diagnosis of adenitis in non-immunocompromised children. OBJECTIVES: To describe the clinical and pathological findings in childhood NTMB adenitis and study the possible usefulness of antimicrobial therapy in addition to surgery. METHODS: We present eight cases of neck lymphadenitis occurring over a 5-year period. All of the children received combined chemotherapy, and six also underwent surgery. Of the two remaining patients, the parents of one child refused surgery and a watchful approach was adopted in the other. RESULTS: Complete clinical recovery was achieved in all patients except one who did not undergo surgery. CONCLUSIONS: Prolonged administration of two antibiotics (of which one must be clarithromycin) in addition to surgery was well-tolerated and could be useful in patients with NTMB neck lymphadenitis.

Polymerase chain reaction for rapid diagnosis of tuberculous meningitis in AIDS patients.

We used a test based on the polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis DNA in 11 CSF samples from 10 HIV-seropositive patients in whom tuberculous meningitis was suspected. PCR was positive in nine samples from eight patients in whom clinical data, CSF findings, evidence of tuberculosis at an extraneural site, and response to antituberculous drug therapy supported the diagnosis, whereas cultures were positive in five of these nine samples and staining in only one. The PCR was negative in 14 CSF samples from the control group.

A review of peripheral tuberculous arthritis.

There is a clear difference between TB infection and TB. Transition from the former to the latter involves host factors and perhaps environmental elements. Currently, more individuals with immunosuppressive situations caused by aging, debilitating diseases, immunosuppressive therapies, and HIV-III infection are predisposed to secondary forms of TB. Different patterns of clinical presentation, at variance with those previously described, could result from these changes in the host. PTBA is a good example. In the present review of 52 patients, we found that patient age is rising, as has been reported in other western countries. We also found that oligoarthritis and involvement of non-weight-bearing joints is becoming more common. From the diagnostic point of view, histological studies and cultures of synovial tissue remain the most reliable tests. Awareness of these factors, recognition of changing patterns, proper use of diagnostic procedures, and early treatment should improve the outcome of patients.

Evaluation of an immunohistochemical test with polyclonal antibodies raised against mycobacteria used in formalin-fixed tissue compared with mycobacterial specific culture.

An immunohistochemical (IH) test (commercially available polyclonal antiserum rabbit anti-Myco-bacterium bovis; DAKO A/S) was used to detect the presence of mycobacteria in 65 formalin-fixed, paraffin-embedded tissue blocks from different organs, showing necrotizing caseous granuloma lesions on hematoxylin and eosin sections from 65 patients. These 65 samples were dyed using an acid-fast fluorescent technique and compared using the immunohistochemical method. Both results were also compared with the mycobacterial cultures. The IH test, compared with the culture, showed a sensitivity (S) of 52%, a specificity (Sp) of 76%, a positive predictive value (PV pos) of 61% and a negative predictive value (PV neg) of 69%. We analyze these data and discuss the possible causes of false-positive and -negative results of the IH test. This rapid test on paraffin embedded tissue seems valuable in the period when waiting for the culture results.

Recent transmission of tuberculosis in Madrid: application of capture-recapture analysis to conventional and molecular epidemiology.

BACKGROUND: Population-based studies using a combination of molecular techniques and conventional epidemiological methods have been used to study the dynamics of tuberculosis (TB) transmission but the relative utility of each technique has not yet been established. METHODS: A prospective population-based molecular and epidemiological study of patients diagnosed with TB was conducted in three urban districts of Madrid (Spain) during 1997-1999. Analysis was performed using the capture-recapture method including covariates in which conventional epidemiological data and the information on clustered cases obtained by DNA fingerprinting were regarded as independent and complementary procedures. RESULTS: The estimate obtained by molecular analysis alone, that 31.6% of TB cases were due to recent transmission, was revised to 44.8% (95% CI: 31.4-58.2) using the capture-recapture method. The estimated completeness of the combined databases for identification of recent transmission was 59.2%. Underestimation of the true prevalence of recent transmission was higher with conventional epidemiology than molecular analysis, particularly for patients <35 years old and those with a history of imprisonment. CONCLUSIONS: In this study, use of the capture-recapture technique allowed us to combine epidemiological information obtained by conventional and molecular methods to quantify the number of cases of recently transmitted TB in the community and identify specific populations at high risk of disease. This information is clearly important because such groups are a prime target for improved TB control measures. In the long term, this combination of techniques may contribute significantly to control the spread of TB.

Mycobacterium fortuitum complex endocarditis-case report and literature review.

Endocarditis due to Mycobacterium fortuitum complex is a rare entity generally linked to the hospital environment. Only 18 cases have been published since 1966. Here we present a case of a female who developed an endocarditis due to Mycobacterium chelonae after valve replacement as well as a review of the literature. The course of this kind of endocarditis is generally subacute and the outcome is usually fatal. Blood cultures were positive in 75% of cases of metallic valve endocarditis, versus 20% in bioprostheses. The treatment must include antibiotics that have shown activity against these mycobacteria, such as amikacin, imipenem, cefoxitin, fluorinated quinolones and macrolides (especially clarithromycin). Surgical removal is recommended. Although the prognosis for the patient is poor, we should expect better outcomes with the use of new antibiotic regimens.

The influence of HIV infection and imprisonment on dissemination of Mycobacterium tuberculosis in a large Spanish city.

SETTING: Culture-positive cases of tuberculosis (TB) from the urban population of southern Madrid and from all the prisons located throughout the city. OBJECTIVE: To determine the frequency with which common strains of Mycobacterium tuberculosis cause disease among patients from both the urban and prison populations of a large Spanish city. DESIGN: Restriction fragment length polymorphism analysis was performed on culture-positive cases of TB identified between 1 January 1997 and 31 December 1998. Risk factors that might be associated with the dissemination of common strains of TB among the two populations were also investigated. RESULTS: Two hundred and twenty-one cases of culture-positive TB were identified, 99 (47.8%) of which were grouped in 23 clusters. Eleven were general clusters that spanned the prison and urban populations involved 69 patients (31.2%). Univariate analysis of risk factors showed that age <35 years, human immunodeficiency virus (HIV) infection, intravenous drug use and current or previous imprisonment were all associated at a statistically significant level with inclusion in general clusters. The final logistic regression model showed an interaction between HIV infection and incarceration. CONCLUSIONS: Dissemination of common strains of M. tuberculosis between prison inmates and the urban population of Madrid is significant, and involves subjects with a history of imprisonment and HIV infection.

Paradoxical responses in a cohort of HIV-1-infected patients with mycobacterial disease.

SETTING: Paradoxical worsening or relapse of opportunistic infections has been described after initiation of highly active anti-retroviral therapy (HAART) in human immunodeficiency virus (HIV) infected patients. DESIGN: Retrospective study of a group of 33 HIV-infected patients with mycobacterial disease analysing the incidence and characteristics of patients with and without paradoxical response after starting HAART and/or mycobacterial treatment. RESULTS: Nine patients in the group had paradoxical response. No significant difference of baseline characteristics was observed in these patients. The decrease in viral load was significantly greater among patients with paradoxical response than in patients without. CONCLUSION: No clinical difference was found in the evolution of HIV-infected patients with mycobacterial disease after the resolution of the episode of paradoxical response.

Cutaneous infection with rapidly growing mycobacteria in patients with systemic rheumatic disease.

We report two patients with systemic rheumatic disease being treated with steroids whose cases were complicated by subcutaneous nodules. In both, clinical and histological features suggested cutaneous infection and M. chelonae was isolated from skin specimens. Antibiotic therapy in both and surgery in one led to healing after a prolonged course. A review of the literature and our experience with these two patients suggest that rheumatic patients on steroid therapy are at risk of infection with these unusual pathogens. Knowledge of the risk factors and the distinctive picture of cutaneous mycobacteriosis should improve its diagnosis and therapy.

Evaluation of the detection of Mycobacterium tuberculosis with metabolic activity in culture-negative human clinical samples.

Mycobacterium tuberculosis is assumed to remain in a quiescent state during latent infection, being unable to grow in culture. The aim of this study was to evaluate the detection of viable but non-cultivable bacilli with metabolic activity in human clinical samples using a procedure that is independent of the immunological status of the patient. The study was performed on 66 human clinical samples, from patients subjected to routine diagnosis to rule out a mycobacterial infection. Specimens from pulmonary and extra-pulmonary origins were verified to contain human DNA before testing for M. tuberculosis DNA, rRNA and transient RNA by real-time quantitative PCR. Clinical records of 55 patients were also reviewed. We were able to detect viable but non-cultivable bacilli with a metabolic activity in both pulmonary and extra-pulmonary samples. Mycobacterium tuberculosis RNA was detected in the majority of culture-positive samples whereas it was detected in one-third of culture-negative samples, 20% of them showed metabolic activity. Amplifications of the ftsZ gene and particularly of the main promoter of the ribosomal operon rrnA, namely PCL1, seem to be good targets to detect active bacilli putatively involved in latent infection. Moreover, this last target would provide information on the basal metabolic activity of the bacilli detected.

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.

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.

Detection of Mycobacterium tuberculosis DNA in clinical samples by using a simple lysis method and polymerase chain reaction.

We have evaluated the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples from patients with tuberculous infection. Two simple methods for mycobacterial DNA release have been compared: sonication and lysis with nonionic detergents and proteinase K. The more effective method was the enzymatic technique. By using this protocol with 75 specimens we detected M. tuberculosis DNA in all of the samples, whereas only 48 and 71 samples were positive by acid-fast staining and culture, respectively.

In vitro susceptibility of Brucella melitensis to new cephalosporins crossing the blood-brain barrier.

The in vitro susceptibilities of 83 clinical isolates of Brucella melitensis to seven cephalosporins and a monobactam were determined. Ceftizoxime, ceftriaxone, and cefotaxime were the most effective agents tested, with MICs ranging from 0.25 to 2 micrograms/ml. Moxalactam, cefoperazone, cefuroxime, and ceftazidime showed MICs between 4 and 64 micrograms/ml, with moxalactam being the most active agent in this group. Aztreonam showed poor activity, with MICs higher than 64 micrograms/ml.

[Comparison of the Bacter system and Löwenstein-Jensen medium for cultivation of Mycobacterium tuberculosis in clinical samples]. / Comparación del sistema Bactec y medio de Löwenstein-Jensen para el cultivo de Mycobacterium tuberculosis en muestras clínicas.

Over a period of two years, 1364 clinical samples from pulmonary sources, were studied in order to compare the Bactec-TB system with the Löwenstein-Jensen medium, for isolation of mycobacteria. Within this period of time, 93 strains of Mycobacterium tuberculosis were isolated, 71 (76%) were isolated by the Bactec system, and 69 (74%) were isolated by the Löwenstein-Jensen. The number of contaminations was lower for the Bactec system (3.2%) than for the Löwenstein-Jensen medium (5.3%). The mean time of isolation with the Bactec was 16.4 days. The mean time of isolation with Löwenstein-Jensen medium was 21.9 days. Identification of the strains with the Bactec system by the p-nitro-a-acetilamin-beta-hidroxipropiphenone inhibition test gave a 100% correlation with the conventional biochemical methods used for the identification of the same strains growth of Löwenstein-Jensen.