Morphological manifestations of the atypical mycobacteriosis caused by nontuberculous mycobacteria in the HIV infected patients.

Infection with atypical mycobacteria (MAC) is a well-known complication of AIDS that typically occurs only in people with advanced immunodeficiency. We studied tissues from 13 patients with HIV and atypical mycobacterial infection who died in St Petersburg Russia from 2009-2012. Three patterns of disease were identified that suggest effects of host resistance. The first pattern was in people paucibacillary disease. They had positive blood cultures and histologic changes consistent with mycobacterial infection, but no stainable acid fast bacilli (AFB). The second group had disseminated infection in many organs including the lungs with extensive necrosis with many AFB. Finally, the third group had massive infection of many organs, but not the lungs, and only minimal necrosis. These observations suggest significant heterogeneity in atypical mycobacterial infections.

Real-time PCR assay for rapid detection of epidemiologically and clinically significant Mycobacterium tuberculosis Beijing genotype isolates.

Mycobacterium tuberculosis Beijing genotype strains are rapidly disseminating, frequently hypervirulent, and multidrug resistant. Here, we describe a method for their rapid detection by real-time PCR that targets the specific IS6110 insertion in the dnaA-dnaN genome region. The method was evaluated with a geographically and genetically diverse collection representing areas in East Asia and the former Soviet Union in which the Beijing genotype is endemic and epidemic (i.e., major foci of its global propagation) and with clinical specimens.

Unusual large-scale chromosomal rearrangements in Mycobacterium tuberculosis Beijing B0/W148 cluster isolates.

The Mycobacterium tuberculosis (MTB) Beijing family isolates are geographically widespread, and there are examples of Beijing isolates that are hypervirulent and associated with drug resistance. One-fourth of Beijing genotype isolates found in Russia belong to the B0/W148 group. The aim of the present study was to investigate features of these endemic strains on a genomic level. Four Russian clinical isolates of this group were sequenced, and the data obtained was compared with published sequences of various MTB strain genomes, including genome of strain W-148 of the same B0/W148 group. The comparison of the W-148 and H37Rv genomes revealed two independent inversions of large segments of the chromosome. The same inversions were found in one of the studied strains after deep sequencing using both the fragment and mate-paired libraries. Additionally, inversions were confirmed by RFLP hybridization analysis. The discovered rearrangements were verified by PCR in all four newly sequenced strains in the study and in four additional strains of the same Beijing B0/W148 group. The other 32 MTB strains from different phylogenetic lineages were tested and revealed no inversions. We suggest that the initial largest inversion changed the orientation of the three megabase (Mb) segment of the chromosome, and the second one occurred in the previously inverted region and partly restored the orientation of the 2.1 Mb inner segment of the region. This is another remarkable example of genomic rearrangements in the MTB in addition to the recently published of large-scale duplications. The described cases suggest that large-scale genomic rearrangements in the currently circulating MTB isolates may occur more frequently than previously considered, and we hope that further studies will help to determine the exact mechanism of such events.

Russian "successful" clone B0/W148 of Mycobacterium tuberculosis Beijing genotype: a multiplex PCR assay for rapid detection and global screening.

We describe a multiplex PCR assay to detect the Mycobacterium tuberculosis Beijing genotype variant B0/W148, which is considered a "successful" clone of M. tuberculosis, widespread in Russia. Specificity and sensitivity of the assay were 100% based on the analysis of a collection of 516 M. tuberculosis isolates of different genotypes and origins. This assay may be used for accurate and simple detection and surveillance of this clinically and epidemiologically important variant of M. tuberculosis.

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.

Emerging multidrug resistant Mycobacterium tuberculosis strains of the Beijing genotype circulating in Russia express a pattern of biological properties associated with enhanced virulence.

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.

Emerging multidrug resistant Mycobacterium tuberculosis strains of the Beijing genotype circulating in Russia express a pattern of biological properties associated with enhanced virulence

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.

At Baltic crossroads: a molecular snapshot of Mycobacterium tuberculosis population diversity in Kaliningrad, Russia.

The Kaliningrad region is the westernmost part of the Russian Federation; it includes an enclave on the Baltic Sea inside the European Union separated from mainland Russia by Lithuania and Poland. The incidence of tuberculosis in Kaliningrad has shown a steady and dramatic increase from 83/100,000 in 2000 to 134/100,000 in 2006; the rate of multidrug-resistant tuberculosis (MDR-tuberculosis) in the Kaliningrad region was reported to be 30.5% among newly diagnosed tuberculosis patients. This study presents a first molecular snapshot of the population diversity of Mycobacterium tuberculosis in this region. A total of 90 drug-resistant and susceptible M. tuberculosis strains from Kaliningrad were subjected to spoligotyping, 12-locus MIRU typing and mutation analysis of the drug resistance genes rpoB and katG. A comparison with international databases showed that the M. tuberculosis population in this region shares a joint pool of strains with the European part of Russia, and also exhibits a certain affinity with those of its northern European neighbours, such as Poland and Germany. Comparison of the genotyping and drug resistance data emphasized that the high prevalence of the MDR Beijing genotype strains is a major cause of the adverse epidemiological situation of MDR-tuberculosis in the Kaliningrad region.

Mycobacterium tuberculosis Beijing genotype in Russia: in search of informative variable-number tandem-repeat loci.

The Beijing genotype is a globally spread lineage of Mycobacterium tuberculosis. In Russia, these strains constitute half of the local population of M. tuberculosis; they are associated with multidrug resistance and show increased transmissibility. Here, we analyzed traditional and new markers for the rapid and simple genotyping of the Beijing strains. A representative sample of 120 Beijing genotype strains was selected from a local IS6110-restriction fragment length (RFLP) database at the St. Petersburg Pasteur Institute. These strains were subjected to variable-number tandem-repeat (VNTR) typing using 24 loci of a newly proposed format and three hypervariable (HV) loci (QUB-3232, VNTR-3820, and VNTR-4120). Ten of the 27 VNTR loci were monomorphic, while five loci, MIRU26, QUB-26, QUB-3232, VNTR-3820, and VNTR-4120, were the most polymorphic (Hunter Gaston index, >0.5). VNTR typing allowed us to differentiate between two large IS6110-RFLP clusters known to be prevalent across the entire country (clusters B0/W148 and A0) and identified in 27 and 23% of strains, respectively, in the Beijing genotype database. The B0/W148 strains were grouped closely in the VNTR dendrogram and could be distinguished by a characteristic signature of the loci MIRU26 and QUB-26. Consequently, this clinically important IS6110-RFLP variant, B0/W148, likely presents a successful clonal group within the M. tuberculosis Beijing lineage that is widespread in Russia. To conclude, the IS6110-RFLP method and VNTR typing using a reduced set of the most polymorphic loci complement each other for the high-resolution epidemiological typing of the M. tuberculosis Beijing genotype strains circulating in or imported from Russia.

Molecular characterization of ofloxacin-resistant Mycobacterium tuberculosis strains from Russia.

In this work, we studied the variation in the gyrA and gyrB genes in ofloxacin- and multidrug-resistant Mycobacterium tuberculosis strains circulating in northwest Russia. Comparison with spoligotyping data suggested that similar to the spread of multidrug-resistant tuberculosis, the spread of fluoroquinolone-resistant tuberculosis in Russia may be due, at least partly, to the prevalence of the Beijing genotype in a local population of M. tuberculosis.

Rapid detection of the Mycobacterium tuberculosis Beijing genotype and its ancient and modern sublineages by IS6110-based inverse PCR.

The Mycobacterium tuberculosis Beijing genotype strains appear to be hypervirulent and associated with multidrug-resistant tuberculosis. Therefore, the development of a both rapid and simple method to detect the M. tuberculosis Beijing genotype is of clinical interest per se. Previously, we described a simple and fast approach to detect the Beijing genotype based on IS6110 inverse-PCR typing. Here, we evaluated this method against a large, diverse, and recent collection of strains. The study sample included 866 M. tuberculosis strains representing but not limited to the regions in Russia, Europe, and East Asia where the Beijing genotype is endemic. Based on a spoligotyping method, 408 strains were identified as Beijing genotypes; they were additionally subdivided into ancient and modern sublineages based on the analysis of the NTF locus. All strains were further subjected to the IS6110-based inverse PCR. All of the Beijing genotype strains were found to have identical two-band (ancient sublineage) or three-band (modern sublineage) profiles that were easily recognizable and distinct from the profiles of the non-Beijing strains. Therefore, we suggest using IS6110-based inverse-PCR typing for the correct identification of the Beijing genotype and its major sublineages. The method is fast and inexpensive and does not require additional experiments but instead is implemented in the routine typing method of M. tuberculosis.

Origin and primary dispersal of the Mycobacterium tuberculosis Beijing genotype: clues from human phylogeography.

We suggest that the evolution of the population structure of microbial pathogens is influenced by that of modern humans. Consequently, the timing of hallmark changes in bacterial genomes within the last 100,000 yr may be attempted by comparison with relevant human migrations. Here, we used a lineage within Mycobacterium tuberculosis, a Beijing genotype, as a model and compared its phylogeography with human demography and Y chromosome-based phylogeography. We hypothesize that two key events shaped the early history of the Beijing genotype: (1) its Upper Palaeolithic origin in the Homo sapiens sapiens K-M9 cluster in Central Asia, and (2) primary Neolithic dispersal of the secondary Beijing NTF::IS6110 lineage by Proto-Sino-Tibetan farmers within east Asia (human O-M214/M122 haplogroup). The independent introductions of the Beijing strains from east Asia to northern Eurasia and South Africa were likely historically recent, whereas their differential dissemination within these areas has been influenced by demographic and climatic factors.

Analysis of the allelic diversity of the mycobacterial interspersed repetitive units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations.

A study set comprised 44 Mycobacterium tuberculosis strains of the Beijing family selected for their representativeness among those previously characterized by IS6110-RFLP and spoligotyping (Northwest Russia, 1997 to 2003). In the present study, these strains were subjected to mycobacterial interspersed repetitive units (MIRU) typing to assess a discriminatory power of the 12-MIRU-loci scheme (P. Supply et al., J. Clin. Microbiol. 39:3563-3571, 2001). The 44 Russian Beijing strains were subdivided into 12 MIRU types with identical profiles: 10 unique strains and two major types shared by 10 and 24 strains. Thus, basically, two distinct sublineages appear to shape the evolution of the Beijing strains in Russia. Most of the MIRU loci were found to be (almost) monomorphic in the Russian Beijing strains; the Hunter-Gaston discriminatory index (HGDI) for all 12 loci taken together was 0.65, whereas MIRU26 (the most variable in our study) showed a moderate level of discrimination (0.49). The results were compared against all available published MIRU profiles of Beijing strains from Russia (3 strains) and other geographic areas (51 strains in total), including South Africa (38 strains), East Asia (7 strains), and the United States (4 strains). A UPGMA (unweighted pair-group method with arithmetic averages)-based tree was constructed. Interestingly, no MIRU types were shared by Russian and South African strains (the two largest samples in this analysis), whereas both major Russian types included also isolates from other locations (United States and/or East Asia). This implies the evolution of the Beijing genotype to be generally strictly clonal, although a possibility of a convergent evolution of the MIRU loci cannot be excluded. We propose a dissemination of the prevailing local Beijing clones to have started earlier in South Africa rather than in Russia since more monomorphic loci were identified in Russian samples than in South African samples (mean HGDI scores, 0.08 versus 0.17). To conclude, we suggest to use a limited number of MIRUs for preliminary subdivision of Beijing strains in Russian (loci 26 + 31), South African (10 + 26 + 39), and global settings (10 + 26 + 39).

Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears.

We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB.

Detection of embB306 mutations in ethambutol-susceptible clinical isolates of Mycobacterium tuberculosis from Northwestern Russia: implications for genotypic resistance testing.

A total of 183 epidemiologically unlinked Mycobacterium tuberculosis isolates collected in the St. Petersburg area of Russia from 1996 to 2001 were screened for alterations in codon 306 of the embB gene; mutations in this codon are reported to confer resistance to ethambutol (EMB). The embB306 mutations were detected in 14 (48.3%) of 29 EMB-resistant strains and, quite surprisingly, in 48 (31.2%) of 154 EMB-susceptible strains. A discrepancy between the results of phenotypic and genotypic EMB resistance tests was restricted to the strains already resistant to other antitubercular (anti-TB) drugs. In particular, 40 (60%) of the 69 EMB-susceptible strains resistant to rifampin, isoniazid, and streptomycin but none of the 43 pansusceptible strains harbored an embB306 mutation. We hypothesize that the phenomenon observed could reflect the presence of a target other than EmbB for the drug in tubercle bacilli; this unknown target could be sensitized and affected, sensu lato, by EMB during treatment with other first-line anti-TB drugs. Comparison with DNA fingerprinting data showed that, irrespectively of the phenotypic susceptibility profiles, 46 (50.6%) of 91 Beijing family strains and 16 (17.4%) of 92 strains of other genotypes had a mutation in embB306.

Molecular characterization of multiple-drug-resistant Mycobacterium tuberculosis isolates from northwestern Russia and analysis of rifampin resistance using RNA/RNA mismatch analysis as compared to the line probe assay and sequencing of the rpoB gene.

This investigation evaluated the potential of RNA/RNA mismatch analysis for the detection of rifampin resistance among 38 multiple-drug-resistant (MDR) isolates of Mycobacterium tuberculosis from northwestern Russia. The results obtained were compared with a commercialized line probe assay and rpoB sequencing, and the genetic diversity of the isolates was also investigated in parallel using spoligotyping and variable number of tandem DNA repeats (VNTR). The mismatch analysis revealed 3 distinct RNA cleavage profiles permitting the subdivision of the strains into mutation groups 1 to 3, the most common being group 1 (28 of 38 isolates) that contained a majority of strains with a TCG531>TTG (Ser->Leu) mutation, followed by group 2 (6 of 38 isolates) characterized by different mutations in the codon CAC526 (His), and group 3 (4 of 38 isolates), all characterized by a GAC516(Asp) mutation. Spoligotyping revealed the Beijing type to be the most prevalent among mismatch group 1 (24 out of 28 strains), suggesting that the most frequent rpoB mutation among the Beijing family in our setting was TCG531 >TTG (Ser->Leu). All the Beijing type isolates were also characterized by a unique VNTR pattern made up of exact tandem repeats (ETR)-A to E of 42435. We conclude that the Beijing genotype constitutes the major family of MDR-TB isolates currently circulating in northwestern Russia, and that the in-house RNA/RNA mismatch analysis may be successfully used for rapid and reliable diagnosis of rifampin-resistant tuberculosis in this setting.

Detection of isoniazid-resistant Mycobacterium tuberculosis strains by a multiplex allele-specific PCR assay targeting katG codon 315 variation.

We describe a simple multiplex allele-specific (MAS)-PCR assay to detect mutations in the second base of the katG gene codon 315, including AGC-->ACC and ACA (Ser-->Thr) substitutions that confer resistance to isoniazid (INH) in Mycobacterium tuberculosis clinical isolates. The 315 ACC allele is found in the majority of Inh(r) strains worldwide, especially in areas with a high incidence of tuberculosis. The 315 ACA allele is characteristic of the New York City multidrug-resistant (MDR) strain W and its progenies in the United States. The mutations in katG315 are revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. Initially optimized on the purified DNA samples, the assay was then tested on crude cell lysates and auramine-stained sputum slide preparations with the same reproducibility and interpretability of profiles generated by agarose gel electrophoresis. The MAS-PCR assay can be used for the detection of resistance to INH in clinical laboratories in regions with a high prevalence of MDR M. tuberculosis strains.

Detection of ethambutol-resistant Mycobacterium tuberculosis strains by multiplex allele-specific PCR assay targeting embB306 mutations.

We describe a multiplex allele-specific (MAS)-PCR assay to detect simultaneously mutations in the first and third bases of the embB gene codon 306ATG. These mutations are known to confer ethambutol (EMB) resistance in the majority of clinical Mycobacterium tuberculosis isolates worldwide. The mutated bases are revealed depending on the presence or absence of the respective indicative fragments amplified from the embB306 wild-type allele. Initially optimized on purified DNA samples, the assay was tested on crude cell lysates and auramine-stained sputum slide DNA preparations with the same reproducibility and interpretability of the generated profiles in agarose gel electrophoresis. Since EMB resistance is generally linked to multiple-drug resistance (MDR), the MAS-PCR assay for EMB resistance detection can be used in clinical laboratory practice in areas with a high prevalence and a high transmission rate of MDR-EMB-resistant tuberculosis.

High prevalence of KatG Ser315Thr substitution among isoniazid-resistant Mycobacterium tuberculosis clinical isolates from northwestern Russia, 1996 to 2001.

A total of 204 isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different patients in the northwestern region of Russia from 1996 to 2001 were screened by a PCR-restriction fragment length polymorphism (RFLP) assay. This assay uses HapII cleavage of an amplified fragment of the katG gene to detect the transversion 315AGC-->ACC (Ser-->Thr), which is associated with INH resistance. This analysis revealed a 93.6% prevalence of the katG S315T mutation in strains from patients with both newly and previously diagnosed cases of tuberculosis (TB). This mutation was not found in any of 57 INH-susceptible isolates included in the study. The specificity of the assay was 100%; all isolates that contained the S315T mutation were classified as resistant by a culture-based susceptibility testing method. The Beijing genotype, defined by IS6110-RFLP analysis and the spacer oligonucleotide typing (spoligotyping) method, was found in 60.3% of the INH-resistant strains studied. The katG S315T shift was more prevalent among Beijing genotype strains than among non-Beijing genotype strains: 97.8 versus 84.6%, respectively, for all isolates, including those from patients with new and previously diagnosed cases, isolated from 1999 to 2001 and 100.0 versus 86.5%, respectively, for isolates from patients with new cases isolated from 1996 to 2001. The design of this PCR-RFLP assay allows the rapid and unambiguous identification of the katG 315ACC mutant allele. The simplicity of the assay permits its implementation into routine practice in clinical microbiology laboratories in regions with a high incidence of TB where this mutation is predominant, including northwestern Russia.