Beijing Family Research Articles

Adhesion to and invasion of pulmonary epithelial cells by the F15/LAM4/KZN and Beijing strains of Mycobacterium tuberculosis

Globally, specific genotypes of Mycobacterium tuberculosis have been shown to dominate in patients, suggesting that these are more successful pathogens. One such genotype, the F15/LAM4/KZN (KZN) family of M. tuberculosis, has predominated in KwaZulu-Natal, South Africa, since the early 1990s. This strain recently evolved from multidrug-resistant to extensively drug-resistant (XDR). The ability of M. tuberculosis strains belonging to the Beijing family, the KZN family, strains with unique DNA fingerprint patterns and laboratory strains (H37Rv and H37Ra) to adhere to and invade a human alveolar (A549) and a human bronchial (BBM) epithelial cell line was investigated. All strains displayed greater adhesion to and invasion of A549 cells as compared to BBM cells. The Beijing and KZN strains combined showed greater adhesion (28 %) than the unique strains (5 %) (P <0.05). The XDR variant of KZN invaded A549 cells more effectively than the other isolates. These results suggest that the successful spread of the Beijing and KZN strains might be related to their interaction with alveolar epithelium.

IS6110-Restriction Fragment Length Polymorphism and Spoligotyping Analysis ofMycobacterium tuberculosisClinical Isolates for Investigating Epidemiologic Distribution in Korea

The Beijing family of Mycobacterium tuberculosis has been emerging in the world. However, there are few nationwide data of genotypic distribution in Korea. This study aimed to identify the genotypic diversity of clinical isolates of M. tuberculosis and to demonstrate the population of Beijing family in Korea. We collected 96 clinical M. tuberculosis isolates from 11 university hospitals nationwide in Korea from 2008 to 2009. We observed 24 clusters in IS6110-RFLP analysis and 19 patterns in spoligotyping. Seventy-five isolates were confirmed to be Beijing family. Two isolates of the K strain and 12 isolates of the K family strain were also found. We found that drug resistance phenotypes were more strongly associated with Beijing family than non-Beijing family (P=0.003). This study gives an overview of the distribution of genotypes of M. tuberculosis in Korea. These findings indicate that we have to pay more attention to control of M. tuberculosis strains associated with the Beijing family.

Tuberculosis in the 21st century: an emerging pandemic?

Since the mid-1980ies the world has witnessed a dramatic increase in tuberculosis. Our knowledge of tuberculosis epidemiology was established during the previous epidemic that, at least in Europe, took two hundred years from rise to fall. It is difficult to understand the new epidemic if we assume that the genetics of the host-parasite relationship is unchanged from the previous epidemic.The paper discusses how both host and parasite genetics may have changed. Molecular epidemiology done in Archangel, Russia, where most of the classical reasons for increase in tuberculosis were absent, indicated that strains of Mycobacterium tuberculosis with changed biological properties could be responsible. Strains belonging to the so-called Beijing family were strongly associated with primary drug resistance and caused clusters ten times as big as “traditional” strains.The paper describes how the research consortium takes these observations further to explore the importance of “new” strains of tubercle bacilli in the on-going pandemic. What are the changes in biological properties and what genetic changes do they reflect? The previous epidemic may have changed the genetic susceptibility of the human host by selection, but what has constituted a selective pressure for the bacillary population? The two most dramatic changes in the environment of M. tuberculosis are mass introduction of chemotherapy and BCG vaccination. We explore further the nature of drug resistance in these strains and the possibility that BCG may fail to protect against bacilli of the new pandemic.Lastly the paper points at some action that can be taken instantly and that may have a major impact on transmission, even before the questions mentioned above are answered. The clue here is to shorten the time of transmission by a rapid test to secure early diagnosis and treatment. What is needed, however, is not a diagnostic test for tuberculosis, but a simple screening test with high sensitivity that could tell us whom among the numerous people who have a chronic cough and systemic symptoms who are the true “tuberculosis suspects” eligible for rapid examination of sputum by smear microscopy

Strain Typing of Mycobacterium tuberculosis Isolates from Korea by Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats

Mycobacterium tuberculosis is one of the most clinically significant infectious agents. Especially during mass outbreaks, accurate identification and monitoring are required. The proportion of Beijing family members is very high among infecting strains, and spoligotyping is not suitable for strain typing. Therefore, we studied the homogeneity of isolates using the mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) method and identified its utility for carrying out molecular epidemiologic analysis. Eighty-one clinical M. tuberculosis isolates that had previously been analyzed by spoligotyping were used in this study. We used the 12 standard MIRU loci and further four exact tandem repeat (ETR) loci (ETR-A, -B, -C, and -F). Four strains each of randomly selected Beijing and Beijing-like families were subjected to IS6110- restriction fragment length polymorphism analysis. All 81 samples showed amplification products of all VNTR loci, and all of them showed differences in at least one locus. The calculation of the Hunter-Gaston diversity index (HGDI) for MIRU-VNTR gave the value of 0.965. Discriminatory index in the six loci (MIRU-10, -16, -26, -31, -39, and ETR-F) were found to be highly discriminated (HGDI >0.6). Beijing and Beijing-like family isolates were discriminated into different MIRU-VNTR types. MIRU-VNTR analysis by using well-selected loci can be useful in discriminating the clinical M. tuberculosis isolates in areas where the Beijing family is predominant.

P238 Identification of Beijing strain of Mycobacterium tuberculosis super family in pulmonary tuberculosis patients with culture positive specimens with multiplex PCR

A selection of genetic markers was used to study the evolution of Mycobacterium tuberculosis Beijing family strains in northwestern Russia. A total of 221 of 434 epidemiologically unlinked isolates studied in 1996–2001 belonged to the Beijing family as determined by standard spoligotyping (signals 35–43). Ninety-six percent of these Beijing isolates (“typical”) were closely related in IS6110-RFLP (D > 0.85) while 9 remaining isolates (2 different profiles, “atypical”) were more distant from the rest (D = 0.6–0.7). Further analysis was performed on a selection of 12 typical and both atypical Beijing strains with different IS6110-RFLP profiles (2 isolates each). All 28 Beijing isolates studied had the KatG 463Leu allele, an intact mtp40 fragment of the mpcA gene, and an identical structure of the DR locus (15 DVRs) with an upstream IS6110 copy in opposite orientation. The IS6110-RFLP based neighbor-joining (distance) and quartet-puzzling (maximum-likelihood) trees showed that the branch lengths were considerably longer for atypical Beijing strains. Typical Beijing strains had the 1.02 kb Rv3135 PPE-family gene and two IS1547 copies (iplA and iplB), one of them (iplB) disrupted by IS6110 insertion. Atypical Beijing strains had the 1.97 kb Rv3135 gene and a single intact IS1547/iplA copy. We suggest that the M. tuberculosis Beijing family strains currently circulating in the northwest of Russia are relatively ancient and thus appear to be endemic in this region since evolutionarily distant time. The prevalent typical Beijing strains (96%) are likely to be of monophyletic origin and their ongoing dissemination has started recently: these strains differ in rapidly evolving IS6110-RFLP but have identical structure of other polymorphic genome regions studied. The atypical Beijing strains (4%) are evolutionary older; they probably had a common (unknown) ancestor with typical Beijing strains.

Allelic diversity of variable number of tandem repeats provides phylogenetic clues regarding the Mycobacterium tuberculosis Beijing family

The Beijing family is the putative hypervirulent lineage of Mycobacterium tuberculosis that has been endemic in East Asia and has disseminated worldwide. The genetic population structure of Beijing family strains with regard to Japan is notable in its high diversity and dominance of the ancestral sublineage, in contrast to the modern sublineage found worldwide. Therefore, it is expected to be a suitable population model for investigating the microevolutionary process of the lineage. Variable number of tandem repeats (VNTR) has become a reliable genotyping method for M. tuberculosis, but its dynamics in the phylogenetic process remains unclear. Using 355 clinical Beijing family isolates in Japan, genetic traits, including VNTR, were analyzed and subjected to minimum spanning tree (MST) reconstruction. In the results, the topology of the tree was tightly related to other genotypic characters. We also found that some VNTR alleles were specific to each sublineage and provided clues for reconstructing a valid MST topology for the population. It is suggested that VNTR typing can elucidate genetic markers representing the phylogenetic classification in the lineage.

Characterization of Mycobacterium tuberculosis complex isolated from iranian and afghani patients by spoligotyping method

Designing newer drugs, vaccines, and diagnostic techniques is dependent on better understanding of M. tuberculosis virulence mechanism. In this study the prevalence of pcaA gene was determined in M. tuberculosis strains typed by spoligotyping. The associated risk factors among patients with different nationalities residing in Iran were also determined. The isolated M. tuberculosis strains have been characterized by performing susceptibility tests against four first-line antituberculosis drugs and were then subjected to spoligotyping characterization. PCR was used for detection of pcaA gene and its nucleotide sequence was also determined. Spoligotyping of M. tuberculosis strains resulted in 140 different patterns. One hundred twenty two (87.1%) of these spoligotype isolates were unique and reported for the first time. The remaining18 (12.8%) spoligotype patterns were previously reported from other geographical regions of the world. Haarlem family was most prevalent than other genotype. Antibiotic resistances were higher in those isolated from the Iranian patients. The pcaA gene was detected in M. tuberculosis clinical isolates but not in saprophyte strains such as M. kansasi. The results showed that, spread of M. tuberculosis strains belonging to the Beijing family among Iranian patients has to be considered seriously. This study confirmed the widespread existence of pcaA gene in almost all the clinical isolates. It is also important to undertake studies to identify which factors are the most significant to consider in tuberculosis control program.

Characterization of Mycobacterium tuberculosis complex isolated from iranian and afghani patients by spoligotyping method

Designing newer drugs, vaccines, and diagnostic techniques is dependent on better understanding of M. tuberculosis virulence mechanism. In this study the prevalence of pcaA gene was determined in M. tuberculosis strains typed by spoligotyping. The associated risk factors among patients with different nationalities residing in Iran were also determined. The isolated M. tuberculosis strains have been characterized by performing susceptibility tests against four first-line antituberculosis drugs and were then subjected to spoligotyping characterization. PCR was used for detection of pcaA gene and its nucleotide sequence was also determined. Spoligotyping of M. tuberculosis strains resulted in 140 different patterns. One hundred twenty two (87.1%) of these spoligotype isolates were unique and reported for the first time. The remaining18 (12.8%) spoligotype patterns were previously reported from other geographical regions of the world. Haarlem family was most prevalent than other genotype. Antibiotic resistances were higher in those isolated from the Iranian patients. The pcaA gene was detected in M. tuberculosis clinical isolates but not in saprophyte strains such as M. kansasi. The results showed that, spread of M. tuberculosis strains belonging to the Beijing family among Iranian patients has to be considered seriously. This study confirmed the widespread existence of pcaA gene in almost all the clinical isolates. It is also important to undertake studies to identify which factors are the most significant to consider in tuberculosis control program.

Induction of Cell Death in Human Macrophages by a Highly Virulent Korean Isolate of Mycobacterium tuberculosis and the Virulent Strain H37Rv

Recent studies have suggested that virulent strains of Mycobacterium tuberculosis induce apoptosis in macrophages less often than do attenuated strains. K-strain, which belongs to the Beijing family, is the most frequently isolated clinical strain of M. tuberculosis in Korea. In this study, we investigated the differential induction of cell death in human monocytic THP-1 cells by K-strain and H37Rv, a virulent but laboratory-adapted strain of M. tuberculosis. Although no significant difference in growth rate was observed between the cells exposed to K-strain and those exposed to H37Rv, the levels of protective cytokines such as tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12p40 were lower in K-strain-infected cells than in H37Rv-infected cells. Cell viability assays showed that both K-strain and H37Rv, but not heat- or streptomycin-killed bacteria, induced THP-1 cell death in a TNF-independent manner. In contrast, double staining with fluorochrome-labelled inhibitors of caspase and propidium iodide and lactate dehydrogenase release assays revealed that K-strain induced significantly higher levels of necrotic cell death, rather than apoptosis, in THP-1 cells than did H37Rv. Anti-apoptotic Bcl-2, Mcl-1, Bfl-1 and Bcl-xL in the cells were significantly upregulated following infection with K-strain compared with H37Rv, whereas Bax was slightly upregulated in response to infection with both H37Rv and K-strain. These results suggest that the highly virulent K-strain keeps cellular apoptosis as a host defense mechanism to a minimum and induces necrosis in macrophages.

Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosisin Taipei

BackgroundThe control of tuberculosis in densely populated cities is complicated by close human-to-human contacts and potential transmission of pathogens from multiple sources. We conducted a molecular epidemiologic analysis of 356 Mycobacterium tuberculosis (MTB) isolates from patients presenting pulmonary tuberculosis in metropolitan Taipei. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture.MethodsA total of 356 isolates were genotyped by standard spoligotyping and the strains were compared with in the international spoligotyping database (SpolDB4). All isolates were also categorized using the 15 loci MIRU-VNTR typing method and combin with NTF locus and RD deletion analyses.ResultsOf 356 isolates spoligotyped, 290 (81.4%) displayed known spoligotypes and 66 were not identified in the database. Major spoligotypes found were Beijing lineages (52.5%), followed by Haarlem lineages (13.5%) and EAI plus EAI-like lineages (11%). When MIRU-VNTR was employed, 140 patterns were identified, including 36 clusters by 252 isolates and 104 unique patterns, and the largest cluster comprised 95 isolates from the Beijing family. The combination of spoligotyping and MIRU-VNTR revealed that 236 (67%) of the 356 isolates were clustered in 43 genotypes. Strains of the Beijing family was more likely to be of modern strain and a higher percentage of multiple drug resistance than other families combined (P = 0.08). Patients infected with Beijing strains were younger than those with other strains (mean 58.7 vs. 64.2, p = 0.02). Moreover, 85.3% of infected persons younger than 25 years had Beijing modern strain, suggesting a possible recent spread in the young population by this family of TB strain in Taipei.ConclusionOur data on MTB genotype in Taipei suggest that MTB infection has not been optimally controlled. Control efforts should be reinforced in view of the high prevalence of the Beijing strain in young population and association with drug resistance.

Genetic diversity of theMycobacterium tuberculosisBeijing family in East Asia revealed through refined population structure analysis

The Beijing/W family is the endemic lineage of Mycobacterium tuberculosis in East Asia: it has disseminated worldwide. To elucidate its genetic diversity in Japan, phylogenetic reconstruction was performed using 403 M. tuberculosis Beijing family clinical isolates. Variable number of tandem repeats analysis revealed the strains from Japan to be dispersed mainly among five subgroups in a phylogenetic tree. Interestingly, the genotypes of the strains from China and Mongolia were restricted mainly to a single branch; they exhibited high clonality. IS6110 insertion in the NTF region was also analyzed. The majority (78.6%) of Japanese isolates belonged to the ancient sublineage. The modern Beijing strains were observed to correspond to the branch containing the foreign strains, although the ancient Beijing strains were dispersed among the tree's other branches. Our results reflect the singular genetic diversity and the epidemiological pattern of Beijing M. tuberculosis in Japan.

Evolutionary pathway of the Beijing lineage of Mycobacterium tuberculosis based on genomic deletions and mutT genes polymorphisms

Among the genotypes that prevail in the modern spectrum of Mycobacterium tuberculosis strains, the Beijing genotype is the one that causes major concern, as it is geographically widespread and it is considered hypervirulent. Comparative genomic studies have shown that Beijing strains have principally evolved through mechanisms of deletion of chromosomal regions, designated regions of difference (RD), and mutations. In this paper, we aimed to determine the evolutionary history of Beijing strains through the analysis of polymorphisms generated by deletions of large specific sequences, i.e., RD105, RD181, RD150, and RD142, and by single nucleotide substitutions in genes mutT4 and mutT2, coding for DNA repair enzymes. Based on the molecular characteristics of a collection of Beijing strains recently isolated in Tuscany, Italy, we propose a phylogenetic reconstruction of the Beijing family. According to our model, the Beijing family evolved from a M. tuberculosis progenitor following deletion of the RD207 region, an event responsible for the loss of spacers 1–34 in the direct repeat (DR) locus. The major lineages of the Beijing family then evolved via subsequent deletions of regions RD105, RD181 and RD150. In the most ancient evolutionary lineages genes mutT4 and mutT2 were in wild type configuration; the mutT4 mutation was acquired subsequent to the RD181 deletion in a progenitor strain that, in turn, gave rise to a sublineage bearing the mutT2 mutation. Within the major branches of the Beijing family, deletion of additional spacers in the DR locus led to evolution of sublineages characterized by different spoligotypes. Our evolutionary model of the Beijing family provides a deeper framework than previously proposed for epidemiologic and phylogenetic studies of circulating M. tuberculosis Beijing strains, thus allowing a more systematic and comprehensive evaluation of the relevance of Beijing strain variability.

Progression to Active Tuberculosis, but Not Transmission, Varies byMycobacterium tuberculosisLineage in The Gambia

There is considerable variability in the outcome of Mycobacterium tuberculosis infection. We hypothesized that Mycobacterium africanum was less likely than M. tuberculosis to transmit and progress to tuberculosis disease. In a cohort study of patients with tuberculosis and their household contacts in The Gambia, we categorized 1808 HIV-negative tuberculosis contacts according to exposure to M. tuberculosis or M. africanum. Positive skin test results indicated transmission, and development of tuberculosis during 2 years of follow-up indicated progression to disease. Transmission rates were similar, but rates of progression to disease were significantly lower in contacts exposed to M. africanum than in those exposed to M. tuberculosis (1.0% vs. 2.9%; hazard ratio [HR], 3.1 [95% confidence interval {CI}, 1.1-8.7]). Within M. tuberculosis sensu stricto, contacts exposed to a Beijing family strain were most likely to progress to disease (5.6%; HR relative to M. africanum, 6.7 [95% CI, 2.0-22]). M. africanum and M. tuberculosis transmit equally well to household contacts, but contacts exposed to M. africanum are less likely to progress to tuberculosis disease than those exposed to M. tuberculosis. The variable rate of progression by lineage suggests that tuberculosis variability matters in clinical settings and should be accounted for in studies evaluating tuberculosis vaccines and treatment regimens for latent tuberculosis infection.

Mycobacterium tuberculosis Population Structures Differ Significantly on Two Indonesian Islands

Comparison of Mycobacterium tuberculosis genotype distributions in different areas might help to find determinants of the emergence of certain genotypes, such as the Beijing family. In this study, M. tuberculosis isolates originating from patients from two Indonesian islands were genotyped, and possible associations with patients' characteristics and drug resistance were explored. A high degree of genetic diversity was observed among the M. tuberculosis strains, and a significant difference was found in the geographical distribution of genotype families. The predominant Beijing genotype family was isolated from 268 of 813 patients from West Java (33.0%) versus 12 of 84 patients from Timor (14.3%) (P = 0.002). Family F (East African-Indian) (33.3%) and family D (Latin American and Mediterranean) (20.0%) were more prevalent in Timor. No significant associations were found between genotype families and age, vaccination with Mycobacterium bovis BCG, previous treatment, disease localization, or drug resistance. Possible explanations for the differences in the geographical distribution of the M. tuberculosis genotypes are discussed.

Promising loci of variable numbers of tandem repeats for typing Beijing family Mycobacterium tuberculosis

We analysed the genotypes of 325 Mycobacterium tuberculosis clinical isolates obtained during 2002 throughout Japan. The genotyping methods included insertion sequence IS6110 RFLP, spoligotyping and variable number of tandem repeat (VNTR) analyses. Clustered isolates revealed by IS6110 RFLP analysis accounted for 18.5 % (60/325) of the isolates. Beijing genotype tuberculosis (TB) accounted for 73.8 % (240/325) of the isolates. Using VNTR, we analysed 35 loci, including 12 standard mycobacterial interspersed repetitive units and 4 exact tandem repeats. The discriminatory power of these 16 loci was low. Using VNTR analyses of the 35 loci, 12 loci (VNTRs 0424, 0960, 1955, 2074, 2163b, 2372, 2996, 3155, 3192, 3336, 4052 and 4156) were selected for the genotyping of Beijing genotype strains. Comparison of the discriminatory power of the 12-locus VNTR [Japan Anti-Tuberculosis Association (JATA)] to that of the 15-locus and 24-locus VNTRs proposed by Supply et al. (2006) showed that our established VNTR system was superior to the reported 15-locus VNTR and had almost equal discriminatory power to the 24-locus VNTR. This 12-locus VNTR (JATA) can therefore be used for TB genotyping in areas where Beijing family strains are dominant.

Knowledge of tuberculosis transmission among recently infected patients in Glasgow

To examine perceptions and understanding of disease causation in tuberculosis patients with few epidemiological links detected by contact tracing. An exploratory qualitative study using semi-structured interviews was undertaken. Patients' beliefs were explored against a background of medical thinking about tuberculosis transmission and the current approach to contact tracing. Interviews were undertaken with patients (n=23) or suitable next of kin (n=3). Study patients were diagnosed with tuberculosis in Glasgow, an urban area of Scotland, between 1997 and 2004. All had a genetically indistinguishable 15-banded IS6110 restriction fragment length polymorphism pattern of the Beijing family of Mycobacterium tuberculosis, suggestive of recently transmitted infection, yet few had epidemiological links detectable as a result of contact tracing (30.8%). Interviewees had varying levels of knowledge, but most believed that tuberculosis was caused by a pathogen, spread by person-to-person contact. Modes of transmission were thought to include airborne transmission, sharing utensils with an infected individual, consumption of contaminated foods/liquids, and exchange of bodily fluids. Prolonged contact was not thought to be required for transmission to occur. Impaired immunity, social factors and environmental factors were believed to enhance the potential for transmission. Patients have complex beliefs about tuberculosis transmission and causation, which do not always mirror those of health professionals. Adopting and implementing an approach to contact identification that is aligned with lay beliefs may result in improved contact tracing outcomes.

Molecular typing of Mycobacterium tuberculosis isolates from a rural area of Kanpur by spoligotyping and mycobacterial interspersed repetitive units (MIRUs) typing

Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated the understanding of epidemiology of tuberculosis (TB). This study was done to characterize prevalent genotypes of M. tuberculosis on a collection of 97 isolates based on spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in rural area of Kanpur, North India. In this area different types of interventions are being undertaken and follow-up studies are progressing. Predominant spoligotypes prevalent in this region belonged to Central Asian–Delhi family (CAS1_Del) (37%), East African–Indian family (11%), T1 family (8%) and Beijing (4%) family. Highly distinct MIRU-VNTR genotypes were obtained. Significant spoligotypes such as Beijing and CAS1_Del type were further divided into subtypes with MIRU-VNTR. This preliminary study reveals that CAS is the most predominant family in this rural area of Kanpur. If confirmed in other areas, this combined approach of molecular typing can be preferably be used as first line tool for studying linkage and transmission dynamics of TB in India.

Utility of mycobacterial interspersed repetitive unit typing for differentiating Mycobacterium tuberculosis isolates in Wuhan, China

Mycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping of Mycobacterium tuberculosis, and may represent a feasible approach to study M. tuberculosis molecular epidemiology. To evaluate the use of MIRU typing in discriminating M. tuberculosis strains, isolates from 105 patients in Wuhan City, China, were genotyped by this method as compared to spoligotyping. MIRU typing identified 55 types that defined 21 clusters and 34 unique isolates. The discriminatory power was high [Hunter-Gaston discriminatory index (HGDI), 0.97]. Spoligotyping showed that 86 (81.9 %) of 105 isolates belonged to the Beijing family genotype. For Beijing family and non-Beijing strains, the discriminatory power of MIRU was high (HGDI, 0.95 and 0.98, respectively). Among the alleles of the MIRU loci for the Beijing family, only locus 26 was highly discriminative, but for non-Beijing strains, loci 10, 16 and 26 were highly discriminative. MIRU typing is a simple and fast method which may be used for preliminary screening of M. tuberculosis isolates in China.

Molecular Characterization of Clinical Isolates ofMycobacterium tuberculosisand Their Association with Phenotypic Virulence in Human Macrophages

Among 125 clinical isolates of Mycobacterium tuberculosis collected in Hong Kong and Shanghai, China, between 2002 and 2004, IS6110 typing revealed that 71 strains (57%) belonged to the Beijing family. The intracellular growth of the strains in human peripheral blood monocyte-derived macrophages was measured ex vivo on days 0, 3, 6, and 10. Among all tested strains, three hypervirulent strains showed significant increases in intracellular growth after 10 days of incubation. With an initial bacterial load of 10(4) CFU, most of the clinical isolates and H37Ra (an avirulent strain) exhibited no intracellular survival on day 10, while the three hypervirulent strains together with H37Rv (a virulent strain) showed on average a two- to fourfold rise in CFU count. These three hypervirulent strains belonging to a non-Beijing family were isolated from patients suffering from tuberculosis meningitis. Cytokines secreted by gamma interferon-activated macrophages were measured daily after challenge with selected strains of M. tuberculosis. The levels of tumor necrosis factor alpha were elevated after 24 h of infection among all strains, but the levels were significantly lower among the three hypervirulent strains, whereas interleukin 10 (IL-10) and IL-12 were not detected. Results were concordant with the differential expression of the corresponding cytokine genes in activated macrophages, as monitored by real-time PCR. Our findings highlighted that these three hypervirulent strains may possess an innate mechanism for escaping host immunity, which accounts for their characteristic virulence in patients presenting with a more severe form of disease.

Evaluation of variable numbers of tandem repeat as molecular epidemiological markers of Mycobacterium tuberculosis in Japan

Using 243 Mycobacterium tuberculosis isolates obtained in 2001 in Osaka City, Japan, the discriminatory power of variable numbers of tandem repeats (VNTRs) of 12 standard mycobacterial interspersed repetitive units (MIRUs) was assessed. The biggest cluster defined by MIRU-VNTRs consisted of 57 (23.5 %) isolates and they belonged to the Beijing family based on spoligotyping. When additional VNTR loci were included in the MIRU-VNTR analysis, the 57 originally clustered strains were further differentiated by the addition of Queen's University Belfast (QUB)-VNTRs, but not exact tandem repeat-VNTR. The allelic diversity of additional VNTR loci such as VNTR 3232 (QUB-3232), VNTR 2163a (QUB-11a), VNTR 2163b (QUB-11b) and VNTR 1982 (QUB-18) was high in the 57 strains. When the 243 M. tuberculosis isolates were analysed using 16-locus VNTR (the 12 standard MIRUs and the 4 QUB loci) and IS6110 RFLP, the respective Hunter-Gaston discriminatory indexes were 0.9966 and 0.9971. The discrimination power of 16-locus VNTR was equal to that of IS6110 RFLP analysis. If appropriate loci are added to the standard MIRU analysis, VNTR genotyping could be a valuable tool for strain typing and epidemiological research of M. tuberculosis in Japan.