Julià González-Martín

[Consensus document on the diagnosis, treatment and prevention of tuberculosis].

Abstract Pulmonary tuberculosis must be suspected in patients with respiratory symptoms longer than 2–3 weeks. Immunosuppression may modify the clinical and radiological presentation. The chest X-ray is highly suggestive of tuberculosis (TB), but is occasionally atypical. The complex radiological tests (CT scan, MRI) are more useful in extrapulmonary TB. At least 3 consecutive representative samples from the clinical location are used for diagnosis, whenever possible. Bacilloscopy and liquid medium cultures are indicated in all cases. Genetic amplification techniques are coadjuvant in moderate or high suspicion of TB. In new cases of TB, administration of isoniazid, rifampin, ethambutol, and pyrazinamide (HREZ) for 2 months and isoniazid plus rifampin for 4 months is recommended. For meningitis cases, treatment should continue for up to 12 months, and up to 9 months in spinal TB with neurological affectation and silicosis. Appropriate adjustments with antiretroviral treatment must be made in HIV patients. Combined therapy is recommended to prevent development of resistance. An antibiogram for first line drugs should be performed in all initial extractions from new patients. Treatment control is one of the most important activities in TB management. The Tuberculin Skin Test (TST) is positive in TB infection when ≥ 5mm, and Interferon-Gamma Release Assays (IGRA) are recommended in combination with TST. The standard treatment schedule for infection is 6 months with isoniazid. In pulmonary TB, respiratory isolation is applied for 3 weeks or until 3 negative bacilloscopy samples are obtained.

Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis clinical isolates in the area of Barcelona

OBJECTIVES To determine the proportion and type of mutations in Mycobacterium tuberculosis isolates resistant to streptomycin, and their relationship with the level of resistance and with the epidemiological molecular pattern of the isolates. METHODS Sixty-nine streptomycin-resistant isolates from a M. tuberculosis strain collection (1995-2005) from Barcelona were studied. The MIC of streptomycin for each isolate was determined using the proportions method with Middlebrook 7H11 medium. The entire rpsL gene and two specific fragments of the rrs gene (the 530 loop and the 912 region) were sequenced. IS6110-restriction fragment length polymorphism and spoligotyping were performed in each isolate. RESULTS Twenty-six (26/69, 37.7%) streptomycin-resistant isolates presented a mutation in either the rpsL gene and/or the rrs530 loop, with no mutation in the rrs912 region. Seventeen (24.6%) isolates showed rpsL mutations (codons 43 and 88) associated with high MIC levels. Nine (13.0%) isolates had alterations in the rrs gene (A513T, A513C and C516T). Nineteen isolates (19/64, 29.7%) were classified into seven clusters (containing 2-5 isolates per cluster). Nineteen different spoligotype patterns were found. All the LAM3 spoligotype isolates (10/67, 14.9%) were associated with a C491T change in the rrs gene, being also observed in all LAM3 streptomycin-susceptible isolates. CONCLUSIONS Mutations in the rpsL and rrs genes were detected in 37.7% of streptomycin-resistant M. tuberculosis isolates. High-level resistance was associated with mutations in the rpsL gene, whereas wild-type isolates showed low MIC levels. The presence of the C491T substitution in the rrs gene in streptomycin-susceptible and -resistant isolates demonstrates that this change is an epidemiological marker associated with LAM3 sublineage.

Métodos de determinación de sensibilidad a los antimicrobianos en micobacterias

Mycobacteria are a large group of microorganisms, multiple species of which are major causes of morbidity and mortality, such as tuberculosis and leprosy. At present, the emergence and spread of multidrug-resistant strains of Mycobacterium tuberculosis complex are one of the most serious health problems worldwide. Furthermore, in contrast to M. tuberculosis and Mycobacterium leprae, non-tuberculous mycobacteria (NTM) are more frequently isolated and, in many cases, treatment is based on drug susceptibility testing. This article is a review of the different methods to determine the in vitro drug susceptibility of M. tuberculosis complex and the most relevant NTM isolates. The molecular techniques currently used for rapid detection of resistance of clinical specimens are also analysed.

Impaired fitness of Mycobacterium tuberculosis resistant isolates in a cell culture model of murine macrophages

OBJECTIVES We analysed the ability of Mycobacterium tuberculosis clinical isolates to penetrate and grow inside murine macrophages as a surrogate of fitness. METHODS Thirty-five drug-resistant and 10 drug-susceptible M. tuberculosis isolates were studied in a murine macrophage model from the J774.2 cell line in a 6 day protocol, performing semi-quantitative counts in Middlebrook 7H11 medium. The mycobacterial penetration index (MPI) after infection and the mycobacterial growth ratio (MGR) inside the macrophages were determined to evaluate the fitness of isolates. RESULTS Isolates with the katG S315T mutation and multidrug-resistant (MDR) isolates had a significantly lower MGR compared with drug-susceptible isolates. The MPI of the isolates with the katG S315T mutation showed a significant decrease compared with the MPI of those without this mutation. A trend to significantly lower values was also observed on comparing the MPI of the MDR isolates with that of the drug-susceptible isolates and the isolates resistant to isoniazid. CONCLUSIONS The isoniazid-resistant and MDR isolates with mutations in the katG gene showed decreased multiplication inside murine macrophages, suggesting a lower fitness of M. tuberculosis with these resistance patterns.

Recomendaciones sobre bioseguridad en el laboratorio de micobacterias y revisión de la normativa

Para la microbiologia clinica, Mycobacterium tuberculosis supone un microorganismo cuya manipulacion es dificil, ya que entrana el riesgo de exponerse a un patogeno de transmision principalmente aerea que requiere una contencion de nivel 3. En estos momentos, la mayoria de los accidentes en el laboratorio de micobacterias puede reducirse mediante la practica de procedimientos microbiologicos adecuados, el uso de dispositivos de contencion y proteccion y un diseno de las instalaciones apropiado. La legislacion actual contempla el grado de responsabilidad de las instituciones sanitarias y del personal del laboratorio, y define especificamente el nivel de bioseguridad requerido para el procesamiento de muestras para micobacterias. El respeto a las recomendaciones en contencion primaria y secundaria, asi como el control sistematico del personal de laboratorio y la elaboracion de planes de actuacion frente a accidentes, contribuyen a minimizar los riesgos de ser infectado y proteger a la comunidad.

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.

Xpert® MTB/RIF: utilidad en el diagnóstico de la tuberculosis y de la resistencia a la rifampicina

The advent of the Xpert® MTB/RIF technique was a revolution in the diagnosis of tuberculosis, especially in areas with high incidence and low resources. It allows the detection of Mycobacterium tuberculosis complex and simultaneously the most common resistance mutations to rifampicin in less than 2h. For respiratory samples the sensitivity is very high, but it decreases for extrapulmonary samples and children. Although it is faster and simpler than conventional methods, it presents some limitations and new and better techniques are needed to reduce the number of cases and deaths caused by tuberculosis. This review aims to assess the scientific evidence around the diagnostic performance of Xpert® MTB/RIF in different types of samples and populations, as well as analyse its strengths and limitations for TB diagnosis.

Diagnóstico microbiológico de las infecciones causadas por el género Mycobacterium

Mycobacterial culture has a high sensitivity and is the test of choice for the microbiological diagnosis of tuberculosis and nontuberculous mycobacterial infections. However, the results of this culture require at least 2-3 weeks to obtain positivity. Staining is rapid and can be used as a complementary study, although its sensitivity is low. Gene amplification tests have an intermediate sensitivity and obtain results in 1-2 days. These last tests are indicated in cases with moderate or high clinical suspicion. In HIV patients with severe immunodeficiency (<200 CD4), lipoarabinomannan antigen detection in urine may be useful. The identification of isolates from positive cultures is essential to evaluate the clinical significance of the culture results and consider the therapeutic options available. At present, there is a wide range of identification techniques available, which provide results within just 1-4 days. The future of diagnostic techniques in tuberculosis and nontuberculous mycobacterial infections lies in greater development of gene amplification techniques and promoting the search for biomarkers which enable a new approach to the diagnosis of these infections.

Dosis de protionamida y PAS. Corrección al «Documento de Consenso sobre diagnóstico, tratamiento y prevención de la tuberculosis»

We would first like to thank those of you who commented on our editorial. The observations were both extremely interesting and cordially expressed. Nevertheless, we would like to clarify a small detail: the title “The exception does not prove the rule” paraphrases the statement “The exception proves that the rule is wrong”, made by Richard P. Feynman in the book entitled “The Meaning of It All”. Three of Dr Feynman’s keynote lectures delivered in 1965 are included in this publication, which, curiously, did not appear until 1998 (Ed. Addison-Wesley), years after the death of the great physicist; apparently he was not fond of committing his ideas to paper. The phrase we quote is a simple statement that contradicts Cicero’s original, and now popularized, declaration. In Feynman’s view, if a rule is established, at least in physics and mathematics, it must always be true; a single exception to the rule proves that it is wrong.