Characterization of Mycobacterium tuberculosis Beijing isolates from the Mediterranean area

BACKGROUND. The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases. RESULTS. Only 1% (N = 26) of the isolates from two population-based studies in Spain corresponded to Beijing strains, most of which were pan-susceptible and from Peruvian and Ecuadorian patients. Restriction fragment length polymorphism typing with the insertion sequence IS6110 identified three small clusters (2-3 cases). Mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-15) offered low discriminatory power, requiring the introduction of five additional loci. A selection of the Beijing isolates identified in the Spanish sample, together with a sample of Beijing strains from Italy, to broaden the analysis context in the Mediterranean area, were assayed in an infection model with THP-1 cells. A wide range of intracellular growth rates was observed with only two isolates showing an increased intracellular replication, in both cases associated with contained production of TNF-alpha. No correlation was observed between virulence and the Beijing phylogenetic group, clustered/orphan status, or resistance. The Beijing strain responsible for extensive spread on Gran Canaria Island was also identified in Madrid, but did not lead to secondary cases and did not show high infectivity in the infection model. CONCLUSIONS. The Beijing lineage in our area is a non-homogeneous family, with only certain highly virulent representatives. The specific characterization of Beijing isolates in different settings could help us to accurately identify the virulent representatives before making general assumptions about this lineage.

Genotyping of Mycobacterium tuberculosis isolates from alow-endemic setting in northwestern state of Paraná in Southern Brazil

The purpose of this study was to provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis in a low-endemic setting in northwestern state of Paraná in Southern Brazil. We employed spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) techniques to genotype M. tuberculosisisolates from patients with pulmonary tuberculosis (TB). The 93 isolates analyzed by spoligotyping were divided into 36 different patterns, 30 of which were described in the SITVIT database. Latin American and Mediterranean, Haarlem and T families were responsible for 26.9%, 17.2% and 11.8% of TB cases, respectively. From the 84 isolates analyzed by MIRU-VNTR, 58 shared a unique pattern and the remaining 26 belonged to nine clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. A combination of MIRU-VNTR and spoligotyping resulted in 85.7% discriminatory power (Hunter-Gaston index = 0.995). Thus, combining spoligotyping and MIRU-VNTR typing proved to be most useful for epidemiological study in this low-endemic setting in Southern Brazil. The current study demonstrated that there is significant diversity in circulating strains in the city of Maringá and the surrounding regions, with no single genotype of M. tuberculosispredominating.

Map of ecological networks for landscape planning

This paper presents a method based on a geographical information system (GIS) to model ecological networks in a fragmented landscape. The ecological networks are generated with the help of a landscape model (which integrate human activities) and with a wildlife dispersal model. The main results are maps which permit the analysis and the understanding of the impact of human activities on wildlife dispersal. Three applications in a study area are presented: ecological networks at the landscape scale, conflicting areas at the farmstead scale and ecological distance between biotopes. These applications show the flexibility of the model and its potential to give information on ecological networks at different planning scales.

Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil

A cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC). A two-step approach was used to distinguish M. bovis from other members of MTC: (i) oxyR pseudogene amplification to detect MTC and, subsequently, (ii) allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5%) SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB) results and the source laboratory were associated (p < 0.05) with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3% and 98.1%, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6% and 100%, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100% consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2% (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85% and 98%, and 94% and 100%, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.

Spoligotyping of clinical Mycobacterium tuberculosis isolates from the state of Minas Gerais, Brazil

We performed spoligotyping on 114 strains of the Mycobacterium tuberculosis (Mtb) complex that had been isolated from patients in Minas Gerais Health Units during 2004. A total of 82/114 (72%) clinical isolates were clustered and 32/114 (28%) were unique. Seven shared types containing nine strains were newly created. A total of nine patterns corresponded to unreported orphan strains, as evaluated against all of the strains recorded in the SITVIT2 proprietary database in the Institut Pasteur de la Guadeloupe. The major clades were composed of isolates that belong to the following genotypes: Latin-America and Mediterranean (63/114, 55.3%) (the ill-defined T superfamily) (12/114, 10.5%), Haarlem (8/114, 7%), X clade (6/114, 5.3%), S clade (3/114, 2.6%) and the East-African Indian and Manu types, each with 1/114 (0.9%) isolates. A considerable number of strains (n = 20, 17.5%) showed patterns that did not fall within any of the previously described major clades. We conclude the bulk of tuberculosis (TB) (92/114, 80.7%) in our location is recent evolutionary strains that belong to the principal genetic groups 2/3. Further studies on epidemiology of TB are required to understand Mtb biodiversity and TB transmission in this region.

A rapid detection of multidrug-resistant Mycobacterium tuberculosis by a nitrate reductase assay on blood agar

The susceptibility of 49 Mycobacterium tuberculosis clinical isolates to isoniazid (INH) and rifampisin (RIF) (28 multi-drug resistant-tuberculosis samples) was determined by a nitrate reductase assay (NRA) on blood agar. Agreement between the NRA and other testing methods was found to be 93.8% for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8%, 94.2%, 86.6% and 97%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4%, 96.4%, 95% and 93.1%. In conclusion, we show here that blood agar can be used effectively for the NRA test.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Lower production of IL-17A and increased susceptibility to Mycobacterium bovis in mice coinfected with Strongyloides venezuelensis

The presence of intestinal helminths can down-regulate the immune response required to control mycobacterial infection. BALB/c mice infected with Mycobacterium bovis following an infection with the intestinal helminth Strongyloides venezuelensis showed reduced interleukin-17A production by lung cells and increased bacterial burden. Also, small granulomas and a high accumulation of cells expressing the inhibitory molecule CTLA-4 were observed in the lung. These data suggest that intestinal helminth infection could have a detrimental effect on the control of tuberculosis (TB) and render coinfected individuals more susceptible to the development of TB.

Clinical data and molecular analysis of Mycobacterium tuberculosis isolates from drug-resistant tuberculosis patients in Goiás, Brazil

Drug resistance is one of the major concerns regarding tuberculosis (TB) infection worldwide because it hampers control of the disease. Understanding the underlying mechanisms responsible for drug resistance development is of the highest importance. To investigate clinical data from drug-resistant TB patients at the Tropical Diseases Hospital, Goiás (GO), Brazil and to evaluate the molecular basis of rifampin (R) and isoniazid (H) resistance in Mycobacterium tuberculosis. Drug susceptibility testing was performed on 124 isolates from 100 patients and 24 isolates displayed resistance to R and/or H. Molecular analysis of drug resistance was performed by partial sequencing of the rpoB and katGgenes and analysis of the inhA promoter region. Similarity analysis of isolates was performed by 15 loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. The molecular basis of drug resistance among the 24 isolates from 16 patients was confirmed in 18 isolates. Different susceptibility profiles among the isolates from the same individual were observed in five patients; using MIRU-VNTR, we have shown that those isolates were not genetically identical, with differences in one to three loci within the 15 analysed loci. Drug-resistant TB in GO is caused by M. tuberculosis strains with mutations in previously described sites of known genes and some patients harbour a mixed phenotype infection as a consequence of a single infective event; however, further and broader investigations are needed to support our findings.

Validation of an immunochromatographic assay kit for the identification of the Mycobacterium tuberculosis complex

The performance of the immunochromatographic assay, SD BIOLINE TB Ag MPT64 RAPID®, was evaluated in Madagascar. Using mouse anti-MPT64 monoclonal antibodies for rapid discrimination between the Mycobacterium tuberculosis complex and nontuberculous mycobacteria, the kit was tested on mycobacteria and other pathogens using conventional methods as the gold standard. The results presented here indicate that this kit has excellent sensitivity (100%) and specificity (100%) compared to standard biochemical detection and can be easily used for the rapid identification of M. tuberculosis complex.

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.

Comparative evaluation under routine conditions of the nitrate reduction assay, the proportion assay and the MGIT 960 assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis

The performance of the nitrate reductase assay (NRA) was compared with the proportion method (PM) on Lowenstein-Jensen medium and the BACTEC MGIT960 assay under routine conditions using 160 clinical isolates of Mycobacterium tuberculosis with a high proportion of resistant strains. The mean time to obtain results was 8.8 days and the overall agreements between NRA and PM and NRA and M960 were 95% and 94%, respectively. NRA was easy to perform and represents a useful tool for the rapid screening of drug-resistant M. tuberculosis strains in low-resource countries.

Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 µg/mL and 2.0-0.03 µg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 µg/mL and the RIF concentration was between 2.0-0.06 µg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.