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.

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.

Tratamiento de la linfadenitis. Réplica / Treatment of lymphadenitis. Response

No disponible

[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.

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

Antecedentes: Las infecciones por micobacterias atípicas (también denominadas no tuberculosas [MNTB] o más recientemente ambientales) están desempeñando en los últimos tiempos un papel preponderante en el diagnóstico diferencial de las adenitis en niños inmunocompetentes. Objetivos: Los objetivos de este estudio han sido describir las características clinicopatológicas de las adenopatías por MNTB y evaluar el posible efecto de la terapia antibiótica asociada a cirugía. Métodos: Se presentan 8 casos recogidos en 5 años, todos ellos con afectación cervical. Todos recibieron antimicrobianos combinados junto a cirugía en seis de los casos. En los dos restantes, en uno existió negativa por parte de los padres del paciente y en otro se mantuvo una actitud expectante. Resultados La evolución fue satisfactoria en todos los pacientes menos uno de los no tratados quirúrgicamente. Conclusiones: La asociación prolongada de dos antibióticos siendo uno de ellos claritromicina junto al tratamiento quirúrgico presenta una adecuada tolerancia y podría ser de utilidad en linfadenitis cervical por MNTB

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

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.

Isolated cervical lymphadenopathy as unique manifestation of Brucellosis.

We report the case of a 42-y-old male with an isolated cervical lymphadenopathy due to Brucella melitensis. The diagnosis was established by isolation of B. melitensis in a lymphatic specimen obtained by fine-needle aspiration and confirmed by serological test results showing high levels of Brucella agglutinins. Mycobacteria-specific cultures were negative. Treatment with streptomycin and doxycycline resulted in complete healing. Exceptionally, and only if the epidemiologic context supports it, brucellosis should be considered in the differential diagnosis of lymphadenopathy.

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.

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.

Characterization of a Mycobacterium intracellulare variant strain by molecular techniques.

This paper describes a Mycobacterium intracellulare variant strain causing an unusual infection. Several isolates obtained from an immunocompromised patient were identified as members of the Mycobacterium avium complex (MAC) by the commercial AccuProbe system and biochemical standard identification. Further molecular approaches were undertaken for a more accurate characterization of the bacteria. Up to seven different genomic sequences were analyzed, ranging from conserved mycobacterial genes such as 16S ribosomal DNA to MAC-specific genes such as mig (macrophage-induced gene). The results obtained identify the isolates as a variant of M. intracellulare, an example of the internal variability described for members of the MAC, particularly within that species. The application of other molecular approaches is recommended for more accurate identification of bacteria described as MAC members.

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.

A point mutation in a domain of gamma interferon receptor 1 provokes severe immunodeficiency.

Gamma interferon (IFN-gamma) and the cellular responses induced by it are essential for controlling mycobacterial infections. Most patients bearing an IFN-gamma receptor ligand-binding chain (IFN-gammaR1) deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome. In this report a new mutation that affects the IFN-gammaR1 ligand-binding domain in a Spanish patient with mycobacterial disseminated infection and multifocal osteomyelitis is characterized. The mutation generates an amino acid change that does not abrogate protein expression on the cellular surface but that severely impairs responses after the binding of IFN-gamma (CD64 and HLA class II induction and tumor necrosis factor alpha and interleukin-12 production). A patient's younger brother, who was also probably homozygous for the mutation, died from meningitis due to Mycobacterium bovis. These findings suggest that a point mutation may be fatal when it affects functionally important domains of the receptor and that the severity is not directly related to a lack of IFN-gamma receptor expression. Future research on these nontruncating mutations will make it possible to develop new therapeutical alternatives in this group 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.