Rapid diagnosis of tuberculosis. Detection of drug resistance mechanisms. / Diagnóstico rápido de la tuberculosis. Detección de mecanismos de resistencia.

Tuberculosis is still a serious public health problem, with 10.8 million new cases and 1.8 million deaths worldwide in 2015. The diversity among members of the Mycobacterium tuberculosis complex, the causal agent of tuberculosis, is conducive to the design of different methods for rapid diagnosis. Mutations in the genes involved in resistance mechanisms enable the bacteria to elude the treatment. We have reviewed the methods for the rapid diagnosis of M. tuberculosis complex and the detection of susceptibility to drugs, both of which are necessary to prevent the onset of new resistance and to establish early, appropriate treatment.

In Vivo IS6110 Profile Changes in a Mycobacterium tuberculosis Strain as Determined by Tracking over 14 Years.

Transposition and homologous recombination of IS6110 appear in Mycobacterium tuberculosis along in vivo sequential infections. These events were checked in different clones of a successful strain, M. tuberculosis Zaragoza, with the focus on a variant in which integration of a copy of IS6110 in the origin of replication (oriC) region occurred.

Single nucleotide polymorphism (SNP) analysis used for the phylogeny of the Mycobacterium tuberculosis complex based on a pyrosequencing assay.

BACKGROUND: Different polymorphisms have been described as markers to classify the lineages of the Mycobacterium tuberculosis complex. The analysis of nine single nucleotide polymorphisms (SNPs) was used to describe seven SNPs cluster groups (SCGs). We attempted to classify those strains that could not been categorized into lineages by the genotyping methods used in the routine testing. RESULTS: The M. tuberculosis complex isolates collected in 2010 in our region were analysed. A new method based on multiplex-PCRs and pyrosequencing to analyse these SNPs was designed. For the pyrosequencing assay nine SNPs that defined the seven SCGs were selected from the literature: 1977, 74092, 105139, 232574, 311613, 913274, 2460626, 3352929 and gyrA95. In addition, SNPs in katG(463), mgtC(182), Ag85C(103) and RD(Rio) deletion were detected. CONCLUSIONS: This work has permitted to achieve a better classification of Aragonian strains into SCGs and in some cases, to assign strains to its certain lineage. Besides, the description of a new pattern shared by two isolates "SCG-6c" reinforces the interest of SNPs to follow the evolution of M. tuberculosis complex.

Analysis of mutations in streptomycin-resistant strains reveals a simple and reliable genetic marker for identification of the Mycobacterium tuberculosis Beijing genotype.

The Mycobacterium tuberculosis pandemic is a major health problem, further complicated by an increasing incidence of drug-resistant isolates and the existence of highly transmissible strains, such as those in the Beijing family. Streptomycin (STR)-resistant M. tuberculosis clinical isolates have been analyzed to look for mutations in the rpsL, rrs, and gidB genes. In addition, the Rv1258c gene, which encodes Tap, an efflux pump that transports STR, has been sequenced. Mutations affecting codons 43 and 88 of the rpsL gene were found in 44.4% of the strains, and 16.7% of the strains carried mutations in the rrs gene, both of which probably contribute to STR resistance. Many strains presented with mutations in the gidB gene, but the implication of those mutations in STR resistance remains unclear. Interestingly, a cytosine nucleotide insertion between positions 580 and 581 (denominated Tap(580)) in the Rv1258c gene has been found in all Beijing isolates included in this study, suggesting that it might be a novel polymorphism specific to the Beijing family of M. tuberculosis. A simple and fast restriction fragment length polymorphism (RFLP)-PCR method for detecting the Tap(580) insertion has been developed and used to screen a collection of 220 DNA samples obtained from cultures of M. tuberculosis isolates and 30 respiratory specimens. In all cases, the Beijing and non-Beijing representative samples were identified correctly. Tap(580) is a novel polymorphism specific to the highly transmissible Beijing family, which allows for fast detection of these strains even at the very early stages of infection.

Global study of IS6110 in a successful Mycobacterium tuberculosis strain: clues for deciphering its behavior and for its rapid detection.

The Mycobacterium tuberculosis insertion sequence IS6110, besides being a very useful tool in molecular epidemiology, seems to have an impact on the biology of bacilli. In the present work, we mapped the 12 points of insertion of IS6110 in the genome of a successful strain named M. tuberculosis Zaragoza (which has been referred to as the MTZ strain). This strain, belonging to principal genetic group 3, caused a large unsuspected tuberculosis outbreak involving 85 patients in Zaragoza, Spain, in 2001 to 2004. The mapping of the points of insertion of IS6110 in the genome of the Zaragoza strain offers clues for a better understanding of the adaptability and virulence of M. tuberculosis. Surprisingly, the presence of one copy of IS6110 was found in Rv2286c, as was recently described for a successful Beijing sublineage. As a result of this analysis, a rapid method for detecting this particular M. tuberculosis strain has been designed.

A whole-genome sequencing study of an X-family tuberculosis outbreak focus on transmission chain along 25 years.

Lineage 4/X-family of Mycobacterium tuberculosis is not very notorious, except for the CDC1551 strain. One strain of this family, named Ara50, caused one of the largest tuberculosis outbreaks of the Aragon region, Spain, during the 1990s and remained until 2018. These X-strains are characterised by high transmissibility and by carrying a low copy number of IS6110 in their genomes. Epidemiological data of the 61 patients consisted of inmates, HIV seropositives, intravenous drug users and the homeless. The application of whole-genome sequencing (WGS) to 36 out of 61 isolates, selected by IS6110-RFLP, allowed to confirm 32 as recent transmissions. We found 10 SNPs in genes considered as virulence factors, five of them specific of this strain. WGS identified three sub-clusters (CLSs). The largest one, sub-CLS 1, included 10 cases. Seven of them shared a SNP in the mce3C gene, considered a virulence factor gene. Sub-CLS 2 involved familiar cases, and no link was known for sub-CLS 3. Finally, the strain showed efficacy in latency as a confirmed epidemiological link was established between two cases, with 6 years of distance in their diagnosis. This outbreak study combined epidemiological and molecular analyses in order to elucidate tuberculosis transmission.

[Peritoneal tuberculosis: a 16-year review in a general hospital]. / Peritonitis tuberculosa: revisión de 16 años en un hospital general.

INTRODUCTION: Peritoneal tuberculosis (PT) is a form of abdominal tuberculosis that accounts for 1%-3% of all cases of tuberculous disease and often manifests with non-specific symptoms. METHODS: Cases of PT diagnosed over a period of 16 years are reviewed and the epidemiological, clinical, diagnostic, and outcome data are analyzed. RESULTS: Among 462 patients with extrapulmonary tuberculosis, PT was documented in 13 patients (2.8%), only one whom was infected by human immunodeficiency virus. In most patients, the diagnosis was established on histological findings. In 5 cases, ascitic fluid culture was positive, and the diagnosis was established by this method alone in 3 of them. A multidrug-resistant Mycobacterium tuberculosis Beijing strain was isolated in one patient. The remaining strains isolated were sensitive to classic antituberculosis drugs. Ten patients were treated with 3 antituberculosis agents and 2 were treated with 4 drugs. Eight patients progressed favorably and 5 died due to complications of their underlying medical conditions. CONCLUSION: Peritoneal tuberculosis can develop without evidence of concomitant tuberculous disease. Histology is usually needed to confirm the diagnosis, and the prognosis is good with prompt treatment.

Investigation of a rapidly spreading tuberculosis outbreak using whole-genome sequencing.

This paper describes the application of whole-genome sequencing (WGS) to investigate an outbreak of Mycobacterium tuberculosis occurring in Aragon, Spain, where strains have been submitted to genotyping since 2004. The responsible outbreak strain appeared in our region first in 2014 and it spread to 14 patients in the following three years. WGS found low variability between the isolates with none of the SNPs differences detected more than once, all of which were attributed to a recent transmission. Although two ambiguous bases linked two cases with those who presented the SNP in the same position, the establishment of a definitive transmission route was not possible. The epidemiological data supported the existence of a super-spreader, probably responsible for the majority of the cases involved since there was a two-year delay in diagnoses among cases. This fact would also help explaining the low variability found. The index case was not identified, possibly because it was not diagnosed in Aragon. In addition WGS characterised the strain as a Linage 4.3.3/LAM family and corroborated the susceptibility to anti-tuberculosis drugs observed by the clinical laboratories. This work shows the need to have epidemiological data to support the genomic data in order to clarify the evolution of tuberculosis outbreaks.

Mycobacterium simiae pulmonary infection unmasked during immune reconstitution in an HIV patient.

Highly active antiretroviral therapy in human immunodeficiency virus (HIV) patients may trigger the onset of immune reconstitution inflammatory syndrome (IRIS). Among HIV patients with IRIS, infections are commonly due to Mycobacterium tuberculosis and nontuberculous mycobacteria. We report the first case in Spain and the second in Europe of Mycobacterium simiae pulmonary infection unmasked during immune reconstitution in an HIV patient.