Rapid detection of Mycobacterium tuberculosis in clinical samples by multiplex polymerase chain reaction (mPCR)

Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1 %) and specificity (89.6 %) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify M. tuberculosis from clinical samples and enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.     

An efficient alternative marker for specific identification of Mycobacterium tuberculosis

Rapid and accurate identification of mycobacteria to the species level is important to provide epidemiological information and to guide the appropriate treatment, especially identification of the Mycobacterium tuberculosis (MTB) which is the leading pathogen causing tuberculosis. The genetic marker named as Mycobacterium tuberculosis specific sequence 90 (mtss90) was screened by a bioinformatics software and verified by a series of experiments. To test its specificity, 266 strains of microorganisms and human cells were used for the mtss90 conventional PCR method. Moreover, the efficiency of mtss90 was evaluated by comparing 16S rDNA (Mycobacterium genus-specific), IS6110 (specific identification of MTB complex), mtp40 (MTB-specific) and PNB/TCH method (traditional bacteriology testing) in Mycobacterium strains. All MTB isolates were mtss90 positive. No amplification was observed from any other tested strains with M. microti as an exception. Compared with the traditional PNB/TCH method, the coincidence rate was 99.1 % (233/235). All of the mtss90 positive strains were IS6110 and 16S rDNA positive, indicating a 100 % coincidence rate (216/216) between mtss90 and these two genetic markers. Additionally, mtss90 had a better specificity than mtp40 in the identification of MTB. Lastly, a real-time PCR diagnostic assay was developed for the rapid identification of MTB. In conclusion, mtss90 may be an efficient alternative marker for species-specific identification of MTB and could be used for the diagnosis of tuberculosis combined with other genetic markers.     

IS6110 Transposition and Evolutionary Scenario of the Direct Repeat Locus in a Group of Closely Related Mycobacterium tuberculosis Strains

In recent years, various polymorphic loci and multicopy insertion elements have been discovered in the Mycobacterium tuberculosis genome, such as the direct repeat (DR) locus, the major polymorphic tandem repeats, the polymorphic GC-rich repetitive sequence, IS6110, and IS1081. These, especially IS6110 and the DR locus, have been widely used as genetic markers to differentiate M. tuberculosis isolates and will continue to be so used, due to the conserved nature of the genome of M. tuberculosis. However, little is known about the processes involved in generating these or of their relative rates of change. Without an understanding of the biological characteristics of these genetic markers, it is difficult to use them to their full extent for understanding the population genetics and epidemiology of M. tuberculosis. To address these points, we identified a cluster of 7 isolates in a collection of 101 clinical isolates and investigated them with various polymorphic genetic markers, which indicated that they were highly related to each other. This cluster provided a model system for the study of IS6110 transposition, evolution at the DR locus, and the effects of these on the determination of evolutionary relationships among M. tuberculosis strains. Our results suggest that IS6110 restriction fragment length polymorphism patterns are useful in grouping closely related isolates together; however, they can be misleading if used for making inferences about the evolutionary relationships between closely related isolates. DNA sequence analysis of the DR loci of these isolates revealed an evolutionary scenario, which, complemented with the information from IS6110, allowed a reconstruction of the evolutionary steps and relationships among these closely related isolates. Loss of the IS6110 copy in the DR locus was noted, and the mechanisms of this loss are discussed.     

A dual molecular beacon approach for fast detection of Mycobacterium tuberculosis

The main objectives of this study were to assess a dual molecular beacon approach for fast detection of Mycobacterium tuberculosis (MT). MT beacon (Tb-B) was designed to target the unique IS6110 (114 bp) and rpoB (215 bp) fragment of the MT (H37Ra) genome, and the two fragments were inserted into the PMD-19T vector after purification, by PCR and sequencing, to construct plasmids. Different dilutions of positive plasmid standards were used for dual molecular beacon RT-PCR of rpoB and IS6110, and standard curves were established.The results show that the dual molecular beacon of rpoB and IS6110 detecting MT was stable (CV is 1.91–2.68 %) with a high amplification efficiency (95.6 %). In addition, the strains of non MT did not generate fluorescence signals, while strains of MT did, indicating that the primers and molecular beacons were specific, and only MT complex was amplified. The linear range was wide (10³–10¹¹ copies/mL), and clinical specimens presenting different bacterial counts can be detected.     

Comparison of Real Time IS6110-PCR,Microscopy, and Culture for Diagnosis of Tuberculous Meningitis in a Cohort of Adult Patients in Indonesia

Background

Bacteriological confirmation of tuberculous (TB) meningitis is difficult. Culture is slow and microscopy has insufficient sensitivity. We evaluated real time PCR targeting insertion sequence IS6110 among 230 consecutive adult patients with subacute meningitis in a referral hospital in Indonesia.

Methods

Cerebrospinal fluid (CSF) samples were examined using microscopy, solid and liquid culture, and real time IS6110-PCR with a fluorescence-labeled probe using DNA extracted from CSF. CSF samples from 40 non-infectious neurology patients were used as negative controls. IS6110-PCR results were linked with clinical and CSF characteristics.

Results

Most patients presented with subacute meningitis, after a median of 14 days of symptoms (range 7–30). After exclusion of cryptococcal and bacterial meningitis, 207 patients were classified as definite or probable TB meningitis; 17.9% with HIV infection. Among this group IS6110-PCR gave the highest positivity rate (68%, 95% CI 62–74%) compared with microscopy of ZN-stained slides (11%, 95% CI 7–15%), and mycobacterial culture using solid (36%, 95% CI 29–42%) and liquid (44%, 95% CI 37–51%) media. IS6110-PCR was positive in 92% of patients with culture-positive and 42% of patients with culture-negative probable TB meningitis. Among culture-negative patients, a positive PCR was associated with a history of TB treatment, a longer duration of illness, a higher CSF cell count and protein, and a lower CSF glucose. IS6110-PCR was negative in all CSF samples from non-meningitis control patients.

Conclusions

Real time IS6110-PCR is a quick, sensitive, and specific test for diagnosing of TB meningitis in this setting. Its performance in other (less-developed) settings needs further study.     

Rapid Detection of Mycobacterium tuberculosis by Recombinase Polymerase Amplification

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39°C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.     

Simple Detection of the IS6110 Sequence of Mycobacterium tuberculosis Complex in Sputum,Based on PCR with Graphene Oxide

Graphene oxide (GO) has proven to be a satisfactory DNA-sensor platform for applications in enzyme-free signal amplification, fluorescence-based amplification, and nanoparticle-based platforms because of its excellent electrical, thermal, and optical properties. In this study, we designed a novel platform for the fluorescence detection of biomolecules, using a fluorescent dye-labeled primer and GO. We applied this system for the detection of the IS6110 insertion sequence of the Mycobacterium tuberculosis complex (MTB) and evaluated its feasibility for use in molecular diagnostics. Fifty-four sputum specimens were collected at our institution from October 2010 to March 2012. To detect MTB in the samples, we performed PCR amplification of the IS6110 DNA sequence using FAM-labeled primers, after which the PCR amplicon was incubated with GO and the fluorescence was measured. The results were compared with those obtained by conventional real-time quantitative PCR (RQ-PCR). The fluorescence intensity observed increased in a concentration-dependent manner with the FAM-labeled IS6110 amplicon. The results of the PCR-GO system for detecting IS6110 DNA were in good agreement with those obtained with conventional RQ-PCR (kappa statistic = 0.925). The PCR-GO system detected MTB DNA in 23 of 25 RQ-PCR-positive sputum samples (92.0%; 95% CI, 75.0–98.0%), but not in 29 of 29 RQ-PCR-negative sputum samples (100%; 95% CI, 88.1–100.0%). These results indicate the utility of the PCR-GO system in molecular diagnostics.     

Genetic Diversity of Mycobacterium tuberculosis from Guadalajara,Mexico and Identification of a Rare Multidrug Resistant Beijing Genotype

Determining the genetic diversity of M. tuberculosis strains allows identification of the distinct Mycobacterium tuberculosis genotypes responsible for tuberculosis in different regions. Several studies have reported the genetic diversity of M. tuberculosis strains in Mexico, but little information is available from the state of Jalisco. Therefore, the aim of this study was to determine the genetic diversity of Mycobacterium tuberculosis clinical isolates from Western Mexico. Sixty-eight M. tuberculosis isolates were tested for susceptibility to first-line drugs using manual Mycobacteria Growth Indicator Tube method and genotyped using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) pattern analyses. Forty-seven (69.1%) isolates were grouped into 10 clusters and 21 isolates displayed single patterns by spoligotyping. Three of the 21 single patterns corresponded to orphan patterns in the SITVITWEB database, and 1 new type that contained 2 isolates was created. The most prevalent lineages were T (38.2%), Haarlem (17.7%), LAM (17.7%), X (7.4%), S (5.9%), EAI (1.5%) and Beijing (1.5%). Six (12.8%) of the clustered isolates were MDR, and type 406 of the Beijing family was among the MDR isolates. Seventeen (26.2%) isolates were grouped into 8 clusters and 48 isolates displayed single patterns by IS6110-RFLP. Combination of IS6110-RFLP and spoligotyping reduced the clustering rate to 20.0%. The results show that T, Haarlem, and LAM are predominant lineages among clinical isolates of M. tuberculosis in Guadalajara, Mexico. Clustering rates indicated low transmission of MDR strains. We detected a rare Beijing genotype, SIT406, which was a highly resistant strain. This is the first report of this Beijing genotype in Latin America.     

Transposition mechanism,molecular characterization and evolution of IS<Emphasis Type="Italic">6110</Emphasis>, the specific evolutionary marker of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> complex

The mycobacterial insertion sequence IS6110 proved crucial in deciphering tuberculosis (TB) transmission dynamics. This sequence was also shown to play an important role in the pathogenicity (transmission ability and/or virulence) of Mycobacterium tuberculosis, the main causative agent of TB in humans. In this study, we explored the usefulness of IS6110 and its potential as a phylogenetic/typing marker. We also analyzed the genetic polymorphism and evolutionary trends (selective pressure) of its transposase-encoding open reading frames (ORFs), A and B, using the maximum likelihood method. Both ORFs evolved chronologically through random single nucleotide polymorphisms. They were subjected to strict purifying selection more tight on orfA, with no evidence of significant recombination events. OrfA proved to have a crucial role in regulating the transpositional process. Several analyses showed that IS6110 acquisition antedated the emergence of the Mycobacterium tuberculosis complex. This original copy of IS6110 element was functionally optimal. In conclusion, this study not only demonstrated the usefulness of IS6110 in terms of phylogenetic and typing purposes and its transpositional mechanism, but also informed the scientific community on its evolutionary history.     

Drug resistance and IS6110‐RFLP patterns of Mycobacterium tuberculosis in patients with recurrent tuberculosis in northern Thailand

The emergence of drug resistant Mycobacterium tuberculosis has become a global threat to tuberculosis (TB) prevention and control efforts. This study aimed to determine the drug resistance profiles and DNA fingerprints of M. tuberculosis strains isolated from patients with relapsed or retreatment pulmonary TB in Chiang Rai province in northern Thailand. Significant differences in multidrug resistance (MDR) (P = 0.025) and resistance to isoniazid (P = 0.025) and rifampin (P = 0.046) between first and second registrations of patients with retreatment TB were found. However, there were no significant differences in resistance to any drugs in patients with relapsed TB. The rate of MDR‐TB strains was 12.2% among new patients at first registration, 22.5% among patients with recurrence who had previously undergone treatment at second registration and 12.5% at third registration. Two retreatment patients whose initial treatment had failed had developed MDR‐TB with resistance to all TB drugs tested, including rifampin, isoniazid, streptomycin and ethambutol. IS6110‐RFLP analysis revealed that 66.7% (10/15 isolates) of MDR‐TB belonged to the Beijing family. In most cases, IS6110‐RFLP patterns of isolates from the same patients were identical in relapse and retreatment groups. However, some pairs of isolates from retreatment patients after treatment failure had non‐identical IS6110‐RFLP patterns. These results suggest that, after failure and default treatment, patients with retreatment tuberculosis have a significantly greater risk of MDR‐TB, isoniazid and rifampin resistance than do other patients.     

IS6110-Mediated Deletions of Wild-Type Chromosomes of Mycobacterium tuberculosis

The ipl locus is a site for the preferential insertion of IS6110 and has been identified as an insertion sequence, IS1547, in its own right. Various deletions around the ipl locus of clinical isolates of Mycobacterium tuberculosis were identified, and these deletions ranged in length from several hundred base pairs up to several kilobase pairs. The most obvious feature shared by these deletions was the presence of an IS6110 copy at the deletion sites, which suggested two possible mechanisms for their occurrence, IS6110 transposition and homologous recombination. To clarify the mechanism, an investigation was conducted; the results suggest that although deletion transpositionally mediated by IS6110 was a possibility, homologous recombination was a more likely one. The implications of such chromosomal rearrangements for the evolution of M. tuberculosis, for IS6110-mediated mutagenesis, and for the development of genetic tools are discussed. The deletion of genomic DNA in isolates of M. tuberculosis has previously been noted at only a few sites. This study examined the deletional loss of genetic material at a new site and suggests that such losses may occur elsewhere too and may be more prevalent than was previously thought. Distinct from the study of laboratory-induced mutations, the detailed analysis of clinical isolates, in combination with knowledge of their evolutionary relationships to each other, gives us the opportunity to study mutational diversity in isolates that have survived in the human host and therefore offers a different perspective on the importance of particular genetic markers in pathogenesis.     

Studies of Polymorphic DNA Fingerprinting and Lipid Pattern of Mycobacterium tuberculosis Patient Isolates in Japan

Strain differentiation by DNA restriction fragment length polymorphism (RFLP) has been used mainly for the epidemiological purpose of Mycobacterium tuberculosis infection. In this study, we tried to connect the molecular and phenotypic characteristics of M. tuberculosis patient isolates by comparing the DNA fingerprints obtained by RFLP using IS6110 and lipid patterns using two-dimensional thin-layer chromatography (2-D TLC) with silica gel, since M. tuberculosis has a lipid-rich cell envelope which contributes to the virulence and immunomodulatory properties. We found that 66 isolates of M. tuberculosis from tuberculosis patients showed that the occurrence of IS6110 varied from 1 to 24 copies. The IS6110 patterns were highly variable among isolates. Fifty different RFLP patterns were observed, and 12 RFLP patterns were shared by two or more strains. By computerized analysis of the RFLP patterns of M. tuberculosis patient isolates, we found that 95% of the isolates fell into seven clusters, from A to G, with at least two isolates in each (> 30% similarity). Among the cellular lipids, the phospholipid composition did not differ by strain, whereas the glycolipid pattern differed markedly. Especially, the relative concentration of cord factor and sulfolipid, both of which were known as virulent factors, varied by strain. The fingerprints of some strains showed an association between the DNA and glycolipid patterns, even though some of the same DNA fingerprint strains showed differences in lipid patterns. Among the patient isolates, M. tuberculosis strain 249 possessed a specific glycolipid with 2-O-methyl-L -rhamnose and L-rhamnose, which is rarely found in other strains. This glycolipid showed serological activity against the sera of tuberculosis patients, even if the reactivity was not as strong as trehalose dimycolate. It also showed the inhibition of phagosome-lysosome fusion in macrophages, suggesting involvement with virulence. These results suggest that RFLP analysis using IS6110 is useful for clustering the human isolates of M. tuberculosis, however, for further strain differentiation on virulence, a lipid analysis provides more information.     

Detection of Mycobacterium ulcerans by the loop mediated isothermal amplification method

Background

Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent.

Methodology

We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR.

Principal Findings

The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used.

Conclusion/Significance

The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU     

Simplified amplified-fragment length polymorphism method for genotyping Mycobacterium tuberculosis isolates

A simplified amplified-fragment length polymorphism (AFLP) method was developed and applied to genotype 52 Mycobacterium tuberculosis isolates. This method can be carried out using only one restriction enzyme (XhoI), one double strand adapter, and one PCR primer. The amounts of DNA and DNA polymerase, and the concentrations of primer and Mg²⁺ in the PCR step were optimized using the Basic Sequential Simplex method. AFLP analysis of the isolates generated a total of 24 differently sized bands ranging from 1537 to 121 bp, and 52 different band patterns, with a minimum of 2 and a maximum of 13 bands. The results were compared with the well-established IS6110 restriction fragment length polymorphism (IS6110-RFLP) typing method, which rendered a total of 32 differently sized bands from 1 to 12 kbp, and 52 different band patterns, with a minimum of 3 and a maximum of 15 bands. Therefore, both genotyping methods showed a discriminatory power of samples of 100%. Nevertheless, pairwise comparisons of the 1326 similarity indexes calculated for both typing methods showed a total absence of correlation between the similarity indexes of the two methods. The simplified AFLP method is expected to be more useful for genotyping M. tuberculosis isolates compared to the IS6110-RFLP method, since the former evaluates genetic variations throughout the M. tuberculosis genome. Furthermore, the relatively rapid and low-cost simplified AFLP method compares favorably to the IS6110-RFLP or conventional AFLP methods, and shows great promise for genotyping M. tuberculosis isolates, especially in developing countries or for preliminary screening.     

First Molecular Epidemiology Study of Mycobacterium tuberculosis in Kiribati

Tuberculosis incidence rates in Kiribati are among the highest in the Western Pacific Region, however the genetic diversity of circulating Mycobacterium tuberculosis complex strains (MTBC) and transmission dynamics are unknown. Here, we analysed MTBC strains isolated from culture positive pulmonary tuberculosis (TB) cases from the main TB referral centre between November 2007 and October 2009. Strain genotyping (IS6110 typing, spoligotyping, 24-loci MIRU-VNTR and SNP typing) was performed and demographic information collected. Among 73 MTBC strains analysed, we identified seven phylogenetic lineages, dominated by Beijing strains (49%). Beijing strains were further differentiated in two main branches, Beijing-A (n = 8) and -B (n = 28), that show distinct genotyping patterns and are characterized by specific deletion profiles (Beijing A: only RD105, RD207 deleted; Beijing B: RD150 and RD181 additionally deleted). Many Kiribati strains (59% based on IS6110 typing of all strains) occurred in clusters, suggesting ongoing local transmission. Beijing-B strains and over-crowded living conditions were associated with strain clustering (likely recent transmission), however little evidence of anti-tuberculous drug resistance was observed. We suggest enhanced case finding amongst close contacts and continued supervised treatment of all identified cases using standard first-line drugs to reduce TB burden in Kiribati. Beijing strains can be subdivided in different principle branches that might be associated with differential spreading patterns in the population.     

Development and Evaluation of a Novel Multicopy-Element-Targeting Triplex PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Feces

The enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused by Mycobacterium avium subsp. paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detect M. avium subsp. paratuberculosis in feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02 (an insertion sequence of M. avium subsp. paratuberculosis present in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10 M. avium subsp. paratuberculosis cells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900 and ISMap02 PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection of M. avium subsp. paratuberculosis in fecal samples from cattle and a very valuable tool to be used in PTB control programs.     

Mycobacterium tuberculosis Population in Northwestern Russia: An Update from Russian-EU/Latvian Border Region

This study aimed to characterize the population structure of Mycobacterium tuberculosis in Pskov oblast in northwestern Russia, to view it in the geographical context, to compare drug resistance properties across major genetic families. Ninety M. tuberculosis strains from tuberculosis (TB) patients, permanent residents in Pskov oblast were subjected to LAM-specific IS6110-PCR and spoligotyping, followed by comparison with SITVITWEB and MIRU-VNTRplus databases. The Beijing genotype (n = 40) was found the most prevalent followed by LAM (n = 18), T (n = 13), Haarlem (n = 10), Ural (n = 5), and Manu2 (n = 1); the family status remained unknown for 3 isolates. The high rate of Beijing genotype and prevalence of LAM family are similar to those in the other Russian settings. A feature specific for M. tuberculosis population in Pskov is a relatively higher rate of Haarlem and T types. Beijing strains were further typed with 12-MIRU (followed by comparison with proprietary global database) and 3 hypervariable loci QUB-3232, VNTR-3820, VNTR-4120. The 12-MIRU typing differentiated 40 Beijing strains into 14 types (HGI = 0.82) while two largest types were M2 (223325153533) prevalent throughout former USSR and M11 (223325173533) prevalent in Russia and East Asia. The use of 3 hypervariable loci increased a discrimination of the Beijing strains (18 profiles, HGI = 0.89). Both major families Beijing and LAM had similar rate of MDR strains (62.5 and 55.6%, respectively) that was significantly higher than in other strains (21.9%; P = 0.001 and 0.03, respectively). The rpoB531 mutations were more frequently found in Beijing strains while LAM drug resistant strains mainly harbored rpoB516 and inhA −15 mutations. Taken together with a high rate of multidrug resistance among Beijing strains from new TB cases (79.3% versus 44.4% in LAM), these findings suggest the critical impact of the Beijing genotype on the current situation with MDR-TB in the Pskov region in northwestern Russia.     

Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop‐mediated isothermal amplification assay

Beijing genotype strains of Mycobacterium tuberculosis are geographically widespread and pose a notorious public health problem, these strains causing outbreaks of multidrug‐resistant tuberculosis (TB); some studies have reported an association with drug resistance. Because the prevalence of Beijing strain has a substantial impact on TB control programs, the availability of a rapid and reliable method for detecting these strains is important for epidemiological monitoring of their circulation. The main methods currently used to identify Beijing genotype strains are IS6110 DNA fingerprinting, spoligotyping and PCR to detect specific deletions such as region of difference (RD)207. More recently, multiplex PCR assay using a Beijing‐specific single nucleotide polymorphism (SNP) has been developed for detecting Beijing lineage strains. However, these methods are time‐consuming and technically demanding. In the present study, a loop‐mediated isothermal amplification (LAMP) assay that allows specific identification of Beijing genotype strain was developed. This Beijing genotype strain‐identifying LAMP assay was performed 214 clinical isolates and the results compared with those of conventional PCR that targeted RD207 and Rv0679c‐targreting multiplex PCR for Beijing lineage identification. LAMP assay showed 100% sensitivity and specificity compared with RD207‐PCR. Furthermore, the sensitivity and specificity were 99.3% and 100%, respectively, compared with Rv0679c‐multiplex PCR. This LAMP assay could be used routinely in local laboratories to monitor the prevalence of the Beijing genotype strain and thereby used to help control the spread of these potentially highly virulent and drug resistant strains.     

Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela

Background

Molecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD).

Results

Clinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised. Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis.

Conclusion

This study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies.     

Evolutionary Trends of the Transposase-Encoding Open Reading Frames A and B (orfA and orfB) of the Mycobacterial IS6110 Insertion Sequence

Background

The IS6110 insertion sequence, a member of the IS3 family of insertion sequences, was found to be specific to the Mycobacterium tuberculosis complex (MTBC). Although IS6110 has been extensively characterized as a transposable genetic marker, the evolutionary history of its own transposase-encoding sequence has not, to the best of our knowledge, been investigated.

Methodology/Principal Findings

Here we explored the evolution of the IS6110 sequence by analysing the genetic variability and the selective forces acting on its transposase-encoding open reading frames (ORFs) A and B (orfA and orfB). For this purpose, we used a strain collection consisting of smooth tubercle bacilli (STB), an early branching lineage of the MTBC, and present-day M. tuberculosis strains representing the full breadth of genetic diversity in Tunisia. In each ORF, we found a major haplotype that dominated over a flat distribution of rare descendent haplotypes, consisting mainly of single- and double-nucleotide variant singletons. The predominant haplotypes consisted of both ancestral and present-day strains, suggesting that IS6110 acquisition predated the emergence of the MTBC. There was no evidence of recombination and both ORFs were subjected to strict purifying selection, as demonstrated by their dN/dS ratios (0.29 and 0.51, respectively), as well as their significantly negative Tajima’s D statistics. Strikingly, the purifying selection acting on orfA proved much more stringent, suggesting its critical role in regulating the transpositional process. Maximum likelihood analyses further excluded any possibility of positive selection acting on single amino acid residues.

Conclusions/Significance

Taken together our data fit with an evolutionary scenario according to which the observed variability pattern of the IS6110 transposase-encoding ORFs is generated mainly through random point mutations that accrued on a functionally optimal IS6110 copy, whose acquisition predated the emergence of the MTBC complex. Background selection acting against deleterious mutations led to an excess of low-frequency variants.