Bacillus Calmette-Guérin strains with defined resistance mutations: a new tool for tuberculosis laboratory quality control.

OBJECTIVE: Laboratory quality control (QC) is essential to assess the reliability of tuberculosis diagnostic testing. To provide safe QC reagents for the detection of drug-resistant Mycobacterium tuberculosis, we generated antibiotic-resistant mycobacterial strains of attenuated virulence (M. bovis bacillus Calmette-Guérin (BCG)). METHODS: Seven mono-resistant BCG strains were developed by introducing resistance-conferring mutations into wild-type BCG strains. Mutations were confirmed by dideoxynucleotide sequencing. Phenotypic resistance was quantified by microbroth dilution to determine the MIC90. The capacity of two commercial tests (GeneXpert TB/RIF and Genotype MTBDRplus) to detect resistance-conferring mutations was evaluated independently. RESULTS: Our panel included BCG strains with mutations in rpoB (S450L, I491F), katG (deletion at AA428), gyrA (D94G), rpsL (K43R) and Rv0678c (S63R). These mutations translated respectively into phenotypic resistance to rifampin (MIC ≥8 mg/L), isoniazid (MIC ≥8 mg/L), moxifloxacin (MIC 4 mg/L) and streptomycin (MIC ≥8 mg/L); the Rv0678c mutant showed decreased susceptibility to both clofazimine (MIC 4 mg/L) and bedaqualine (MIC 1 mg/L). GeneXpert (Cepheid) and Genotype MTBDRplus (Hain Lifesciences) both called the rpoB S450L strain rifampin-resistant and the I491F mutant rifampin-susceptible, as expected based on single nucleotide polymorphism positions. Likewise, MTBDRplus called the novel katG deletion mutant isoniazid susceptible despite phenotypic resistance. CONCLUSION: BCG strains engineered to be mono-resistant to anti-tuberculosis drugs can be used as safe QC reagents for tuberculosis diagnostics and drug susceptibility testing.

Mycobacterium tuberculosis transmission over an 11-year period in a low-incidence, urban setting.

SETTING: Montreal, Canada, has a mean annual tuberculosis (TB) incidence of 9 per 100,000 population, 1996-2007. OBJECTIVE: To characterise potential Mycobacterium tuberculosis transmission by patient subgroups defined by age, sex, birthplace, smear and human immunodeficiency virus status, and to estimate the proportion of cases that resulted from transmission between these patient subgroups. DESIGN: Retrospective study using DNA fingerprinting techniques, with clinical and demographic information from the public health department. Among cases with matching fingerprints, a pulmonary index case was identified. The transmission index was defined as the average number of subsequent TB cases generated directly or indirectly from an index case, and was compared among subgroups, including Haitian immigrants. RESULTS: Compared to non-Haitian foreign-born index cases, Canadian-born index cases were associated with 2.38 times as many (95%CI 1.24-4.58) subsequent cases, while Haitian-born index cases were associated with 3.58 times as many (95%CI 1.74-7.36). Smear-positive index cases were not independently associated with increased transmission. However, middle-aged Canadian-born index patients were associated with a disproportionate number of subsequent cases. CONCLUSION: In Montreal, index patients from several high-risk groups are associated with subsequent transmission. This approach can be applied to other low-incidence settings to identify where targeted interventions could potentially further reduce transmission.

Divergence of immunologic and protective responses of different BCG strains in a murine model.

The ongoing evolution of BCG after its introduction in 1921 resulted in strains that differ genetically and phenotypically. Based on a genomic deletion (Region of Difference 2 or RD2) that occurred between 1927 and 1931, BCG strains can be sub-classified by the presence or absence of RD2. The existence of other mutations that distinguish BCG strains precludes simple comparison of RD2-positive and RD2-negative BCG strains to determine the importance, if any, of RD2 for vaccine protection. In this study, we have compared the RD2-containing BCG Russia, BCG Pasteur (which is a natural mutant for RD2), and BCG Pasteur complemented with RD2-genes Rv1979c-Rv1982 through various in vitro and in vivo assays of immunogenicity and protection. We determined that the presence of RD2 did not affect vaccine persistence, but lead to increased immunogenicity, as measured by ELISpot. Additionally, T-cells from animals immunized with BCG Russia and BCG Pasteur::Rv1979c-82 were more effective at killing Mycobacterium tuberculosis in macrophages than T-cells from animals immunized with BCG Pasteur. In a mouse vaccine-challenge model, the presence of RD2 had no effect on pulmonary TB, as measured by M. tuberculosis burden and degree of histopathology, at 4, 8 or 12 weeks post-infection. The presence of RD2 was however associated with decreased dissemination of M. tuberculosis to the spleen. Together, our data demonstrated that the loss of RD2 resulted in decreased immunogenicity but did not affect protection against pulmonary TB, indicating a dissociation between these phenotypes associated with BCG vaccination.

Use of simulated sputum specimens to estimate the specificity of laboratory-diagnosed tuberculosis.

SETTING: Cross-contamination is not uncommon in mycobacteriology laboratories of high-income countries, as documented by bacterial genotyping. The extent of this problem in low-income countries is largely unknown, where this method is impractical. OBJECTIVE: To estimate the rate of cross-contamination in a high-volume tuberculosis (TB) laboratory in South Africa. DESIGN: Simulated sputum specimens labelled with false names were sent from a TB clinic, interspersed with patient samples, and processed for culture and microscopy. Results were interpreted in the context of the observed proportion of samples with positive microscopy and culture results. RESULTS: With microscopy, 6/190 (3.2%) simulated specimens were positive (estimated specificity = 96.8%). Considering the 881 positive microscopy results in 6093 clinical samples, we extrapolate that 19.3% (95%CI 7.0-42.8) of positive smears were false-positives. On culture, 2/190 (1.1%) of the simulated specimens were positive for Mycobacterium tuberculosis (estimated specificity = 98.9%). Considering the 1862 positive cultures from 6093 clinical samples, we estimate that 2.4% (95%CI 0.3-8.8) of positive cultures were false-positives. CONCLUSION: Simulated specimens offer a simple means of estimating the proportion of false-positive results, providing information on all sources of potential error from the clinic, through the laboratory and to reporting of results.

Global tuberculosis trends: a reflection of changes in tuberculosis control or in population health?

BACKGROUND: Many international organizations are advocating for new funds for tuberculosis (TB) specific interventions. Although this approach should help reduce TB incidence, improvements in population health may also be important. We have analyzed the association between changes in population health and health service indicators, and concomitant changes in TB incidence between 1990 and 2005. METHODS: Country level data on population health and health services, economic and epidemiologic indicators were obtained for 165 countries. Regression methods were used to estimate the association of changes in potential predictors with changes in TB incidence. RESULTS: Improvements in population health and health services are associated with improvements in TB outcomes. In adjusted analyses, each 1 year increase in life expectancy was associated with a 7.8/100,000 decline in TB incidence. A 1/1000 decrease in mortality rate in children aged <5 years and a 1% increase in measles vaccination coverage (serving as a general health services indicator) was associated with approximately a 1/100,000 decrease in TB incidence. In countries with a lower prevalence of human immunodeficiency virus (HIV) infection, a 1% increase in TB treatment success rate was also associated with a 1/100,000 decrease in incidence. CONCLUSION: Investment in improving population health and health services may be as important as targeted strategies for controlling TB.

Use of geographic and genotyping tools to characterise tuberculosis transmission in Montreal.

SETTING: In Canada, tuberculosis (TB) is increasingly an urban health problem. Montreal is Canada's second-largest city and the second most frequent destination for new immigrants and refugees. OBJECTIVES: To detect spatial aggregation of cases, areas of excess incidence and local 'hot spots' of transmission in Montreal. DESIGN: We used residential addresses to geocode active TB cases reported on the Island of Montreal in 1996-2000. After a hot spot analysis suggested two areas of overconcentration, we conducted a spatial scan, with census tracts (population 2500-8000) as the primary unit of analysis and stratification by birthplace. We linked these analyses with genotyping of all available Mycobacterium tuberculosis isolates, using IS6110-RFLP and spoligotyping. RESULTS: We identified four areas of excess incidence among the foreign-born (incidence rate ratios 1.3-4.1, relative to the entire Island) and one such area among the Canadian-born (incidence rate ratio 2.3). There was partial overlap with the two hot spots. Genotyping indicated ongoing transmission among the foreign-born within the largest high-incidence zone. While this zone overlapped the area of high incidence among Canadian-born, genotyping largely excluded transmission between the two groups. CONCLUSIONS: In a city with low overall incidence, spatial and molecular analyses highlighted ongoing local transmission.

Use of genetic distance as a measure of ongoing transmission of Mycobacterium tuberculosis.

The stability of the genotypic marker IS6110, used to define the epidemiology of Mycobacterium tuberculosis, is one of the most important factors influencing the interpretation of DNA fingerprint data. We propose that evolved strains should be considered together with clustered strains to represent chains of ongoing transmission. For the present study we used a large set of fingerprint data for strains collected between 1992 and 1998 from residents of a community with a high incidence of tuberculosis in Cape Town, South Africa. Interstrain genetic distances were calculated by counting the banding pattern mismatches in the IS6110 DNA fingerprints of different isolates. These data demonstrate that the propensity to change by one or two bands is independent of the IS6110 copy number. Hence, the genetic distance between pairs of isolates can be simply expressed as the number of differences in the banding patterns. From this foundation, a data set which identifies newly evolved strains has been generated. Inclusion of these evolved strains into various molecular epidemiological calculations significantly increased the estimate of ongoing transmission in this study setting. The indication is that nearly all cases of tuberculosis in this community are due to ongoing transmission. This has important implications for tuberculosis control, as it indicates that the control measures used at present are unable to reduce the level of transmission. This technique may also be applicable to the study of low-incidence tuberculosis outbreaks as well as the analysis of epidemiological data from other disease epidemics.

Microevolution of the direct repeat region of Mycobacterium tuberculosis: implications for interpretation of spoligotyping data.

The direct repeat (DR) region has been determined to be an important chromosomal domain for studying the evolution of Mycobacterium tuberculosis. Despite this, very little is known about microevolutionary events associated with clonal expansion and how such events influence the interpretation of both restriction fragment length polymorphism (RFLP) and spoligotype data. This study examined the structure of the DR region in three independently evolving lineages of M. tuberculosis with a combination of DR-RFLP, spoligotyping, and partial DNA sequencing. The results show that the duplication of direct variable repeat (DVR) sequences and single-nucleotide polymorphisms is rare; conversely, the deletion of DVR sequences and IS6110-mediated mutation is observed frequently. Deletion of either single or contiguous DVR sequences was observed. The deletion of adjacent DVR sequences occurred in a dependent manner rather than as an accumulation of independent events. Insertion of IS6110 into either the direct repeat or spacer sequences influenced the spoligotype pattern, resulting in apparent deletion of DVR sequences. Homologous recombination between adjacent IS6110 elements led to extensive deletion in the DR region, again demonstrating a dependent evolutionary mechanism. Different isolates from the same strain family and isolates from different strain families were observed to converge to the same spoligotype pattern. In conclusion, the binary data of the spoligotype are unable to provide sufficient information to accurately establish genotypic relationships between certain clinical isolates of M. tuberculosis. This has important implications for molecular epidemiologic strain tracking and for the application of spoligotype data to phylogenetic analysis of M. tuberculosis isolates.

Calculation of the stability of the IS6110 banding pattern in patients with persistent Mycobacterium tuberculosis disease.

The interpretation of molecular epidemiologic data of Mycobacterium tuberculosis infection is dependent on the understanding of the stability and evolutionary characteristics of the DNA fingerprinting marker used to classify clinical isolates. This study investigated the stability of the IS6110 banding pattern in serial tuberculosis isolates collected from patients resident in an area with a high incidence of tuberculosis. Evolutionary changes were observed in 4% of the strains, and a half-life (t(1/2)) of 8.74 years was calculated, assuming a constant rate of change over time. This rate may be composed of a high rate of change seen during the early disease phase (t(1/2) = 0.57 years) and a low rate of change seen in the late disease phase (t(1/2) = 10.69 years). The early rate probably reflects change occurring during active growth prior to therapy, while the low late rate may reflect change occurring during or after treatment. We demonstrate that the calculation of these rates is strongly influenced by the time interval between onset of disease and sputum sampling. These calculations are further complicated by partial replacement of the original strain population, resulting in the sporadic appearance of clonal variants in sputum specimens. Therefore, the true extent of genetic diversity may be underestimated within each host, thereby influencing molecular epidemiological data used to establish transmission chains.

Evolution of the IS6110-based restriction fragment length polymorphism pattern during the transmission of Mycobacterium tuberculosis.

Interpretation of the molecular epidemiological data of Mycobacterium tuberculosis is dependent on the validity of the assumptions that have been made. It is assumed that the IS6110 banding pattern is sufficiently stable to define epidemiological events representing ongoing transmission. However, molecular epidemiological data also support the observation that the IS6110 banding pattern may change over time. Factors affecting this rate may include the nature and duration of disease in a host and the opportunity to experience different host environments during the transmission cycle. To estimate the rate of IS6110 change occurring during the process of transmission, M. tuberculosis isolates from epidemiologically linked patients were genotypically characterized by restriction fragment length polymorphism (RFLP) analysis. The identification of IS6110 banding pattern changes during ongoing transmission suggested that a rate could be estimated. IS6110 change was significantly associated with strains with >5 IS6110 elements (P = 0.013) and was not observed in low-copy-number isolates. The minimum rate of appearance of variant strains was calculated to be 0.14 variant cases per source-case per year. This data suggest that clustering of isolates based on identical RFLP patterns is expected to underestimate transmission in patients infected with high-copy-number isolates. A model based on the rate of appearance of both variant and invariant strains demonstrates that the genotypically defined population structure may change by 18.6% during the study period of approximately 6.5 years. The implications for the use of RFLP data for epidemiologic study are discussed.

Profound T-lymphocytopenia and cryptococcemia in a human immunodeficiency virus-seronegative patient with disseminated tuberculosis.

A 47-year-old human immunodeficiency virus-seronegative West African man who presented in extremis with cachexia, lymphadenopathy, multiple organ dysfunction, and marked T-lymphocytopenia received the diagnosis of disseminated tuberculosis, cryptococcal pneumonia, and cryptococcemia. His subsequent course and our review of the literature suggest that the profound T-lymphocytopenia and ensuing cryptococcal disease were likely attributable to disseminated tuberculosis.

Comparative genomics of BCG vaccines.

Bacille Calmette-Guérin (BCG) vaccines have been given to more people than any other vaccine. They have also probably resulted in as much controversy as any other vaccine. In clinical trials, the efficacy of BCG vaccination against pulmonary TB has been widely variable. At the same time, a number of investigators have observed phenotypic differences between BCG daughter strains, raising the possibility that differences between BCG products may in some way translate into different outcomes. With recent genomic analysis of BCG strains, it has become possible to piece together the molecular events that have resulted in current BCG vaccines. Between the derivation of BCG in 1921 and the lyophilization of BCG Pasteur 1173 in 1961, there have been at least seven genetic events, including deletions, duplications and a single nucleotide polymorphism. The phenotypic relevance of these changes in BCG vaccines remains to be explored.

Correlation between BCG genomics and protective efficacy.

Between the derivation of bacille Calmette-Guérin (BCG) vaccine in 1921 and the lyophilization of BCG daughter strains in the 1960s, a number of clinical trials were performed looking at the protective efficacy of BCG vaccination against tuberculosis. These trials differed from one another in a number of ways: they employed different methodologies for delivering the vaccine and interpreting outcomes; they were performed on populations with different genetic backgrounds and different levels of exposure to environmental Mycobacteria; and, finally, they used different BCG vaccine strains. The results of these trials were estimates of protective efficacy against pulmonary tuberculosis ranging from about 80% to nil. Because of the differences in outcomes and confounding variables, it is difficult to conclude whether differences in interventions alone may have contributed to the remarkably variable results. Analysis of BCG vaccines used in clinical trials suggests a trend towards decreasing efficacy with increased passage in the laboratory; however, trials that used relatively "older" BCG strains were generally performed at different sites than trials which used "younger" BCG strains. Genomic analysis of BCG vaccines demonstrates that during the half-century of ongoing passage of BCG vaccines in vitro there have been numerous genetic changes, comprising single nucleotide polymorphisms, duplications and deletions. The impact of these changes in the BCG genome on the protective efficacy observed in field trials remains to be determined.

Identification of substrains of BCG vaccine using multiplex PCR.

Current methods for determining the identity of substrains of Mycobacterium bovis BCG (BCG) vaccine are labour intensive, or provide only limited substrain differentiation. In this paper we describe a multiplex PCR that distinguishes between M. tuberculosis (TB) and M. bovis and the non-pathogenic BCG strain, and also subdivides the BCG vaccine substrains investigated into seven distinct fingerprints based on six target regions in the DNA. This test is specific, rapid, reproducible and portable and is proposed as a novel test for BCG vaccine control. It offers substantial advantages over the methods currently in use. Using this test we have characterised a number of commercial BCG vaccines.

Rubella susceptibility predicts measles susceptibility: implications for postpartum immunization.

Measles and mumps antibody titers were measured in 262 pregnant women who were either positive (n=128) or negative (n=134) for rubella antibodies. Susceptibility to measles and mumps was detected in 4.6% (12/262) and 7.6% (14/184) of the women, respectively. Of the rubella-susceptible group, 8.2% were also measles susceptible, whereas only 0.8% of the rubella-immune women were measles susceptible. Susceptibility to mumps was evenly divided between rubella-susceptible (7.8%) and rubella-immune (7.4%) groups.

Genotypic determination of Mycobacterium tuberculosis antibiotic resistance using a novel mutation detection method, the branch migration inhibition M. tuberculosis antibiotic resistance test.

A novel method for the detection of any alteration within a defined sequence has recently been demonstrated (A. Lishanski, N. Kurn, and E. F. Ullman, Nucleic Acids Res. 28:E42, 2000; A. Lishanski, Clin. Chem. 46:9, 2000). Essential to this method are the generation of partial duplexes that are capable of forming four-stranded structures and the ability to detect inhibition of branch migration in these structures (I. G. Panyutin and P. Hsieh, J. Mol. Biol. 230:413-424, 1993). Inhibition of branch migration indicates the presence of sequence alteration. This mutation detection method, termed branch migration inhibition (BMI), is suitable for the detection of drug resistance in M. tuberculosis, which is frequently associated with multiple mutations within known genes. We describe the genotypic determination of the rifampin (RMP) and pyrazinamide (PZA) susceptibilities of M. tuberculosis isolates, using BMI coupled with the luminescence oxygen channeling immunoassay (LOCI) (E. F. Ullman et al., Proc. Natl. Acad. Sci. USA 91:5426-5430, 1994). RMP and PZA resistances are associated with multiple mutations within the rpoB and pncA genes, respectively. M. tuberculosis genomic DNA samples prepared from 46 clinical isolates were used for genotypic determination of RMP resistance in a "blind study." Similarly, PZA resistance was determined using genomic DNA samples prepared from 37 clinical isolates. Full agreement of the genotypic and phenotypic determinations of drug susceptibility was demonstrated. RMP susceptibility determination directly from cells of 10 clinical isolates grown in culture was also demonstrated. The genotypic result of only 1 out of 10 isolates did not agree with the phenotypic susceptibility testing result. Sequence analysis of the rpoB gene of this clinical isolate revealed a single base substitution, most likely a silent point mutation. The new BMI-LOCI mutation detection method is a rapid and accurate procedure for the genotypic determination of the RMP and PZA susceptibilities of M. tuberculosis clinical isolates. BMI can also be detected by using commercially available automated enzyme-linked immunosorbent assay plate formats (Lishanski et al., Nucleic Acids Res. 28:E42, 2000).

A point mutation in the mma3 gene is responsible for impaired methoxymycolic acid production in Mycobacterium bovis BCG strains obtained after 1927.

BCG vaccines are substrains of Mycobacterium bovis derived by attenuation in vitro. After the original attenuation (1908 to 1921), BCG strains were maintained by serial propagation in different BCG laboratories (1921 to 1961). As a result, various BCG substrains developed which are now known to differ in a number of genetic and phenotypic properties. However, to date, none of these differences has permitted a direct phenotype-genotype link. Since BCG strains differ in their abilities to synthesize methoxymycolic acids and since recent work has shown that the mma3 gene is responsible for O-methylation of hydroxymycolate precursors to form methoxymycolic acids, we analyzed methoxymycolate production and mma3 gene sequences for a genetically defined collection of BCG strains. We found that BCG strains obtained from the Pasteur Institute in 1927 and earlier produced methoxymycolates in vitro but that those obtained from the Pasteur Institute in 1931 and later all failed to synthesize methoxymycolates, and furthermore, the mma3 sequence of the latter strains differs from that of Mycobacterium tuberculosis H37Rv by a point mutation at bp 293. Site-specific introduction of this guanine-to-adenine mutation into wild-type mma3 (resulting in the replacement of glycine 98 with aspartic acid) eliminated the ability of this enzyme to produce O-methylated mycolic acids when the mutant was cloned in tandem with mma4 into Mycobacterium smegmatis. These findings indicate that a point mutation in mma3 occurred between 1927 and 1931, and that this mutant population became the dominant clone of BCG at the Pasteur Institute.

Transmission of tuberculosis in San Francisco and its association with immigration and ethnicity.

OBJECTIVE: To determine tuberculosis transmission dynamics in San Francisco and its association with country of birth and ethnicity. METHODS: Restriction fragment length polymorphism (RFLP) typing was performed on Mycobacterium tuberculosis isolates from culture-positive pulmonary tuberculosis patients in San Francisco (1991 through 1996), using IS6110 as a probe. Patients were assigned to clusters based on mycobacterial isolates with identical DNA fingerprints. Clusters were assumed to have arisen from recent transmission. A transmission index was defined as the average number of culture-positive pulmonary tuberculosis cases generated by a single source case and calculated for subgroups. RESULTS: The transmission index was higher in US-born (0.59) than in foreign-born groups (0.21), and was highest in blacks, in particular those aged under 35 years. The increased transmission index among blacks was not explained by smear-positivity, human immunodeficiency virus infection, or increased susceptibility to disease progression. CONCLUSION: US-born tuberculosis cases generated more secondary cases than immigrants. Young blacks appear to be a high-risk group for tuberculosis transmission. These results suggest the need to develop interventions targeted towards this risk group.

Genetic distances for the study of infectious disease epidemiology.

Molecular epidemiologic studies of infectious pathogens 1) generate genetic patterns from a collection of microorganisms, 2) compare the degree of similarity among these patterns, and 3) infer from these similarities infectious disease transmission patterns. The authors propose a quantitative approach using genetic distances to study the degree of similarity between patterns. Benefits of such genetic distance calculations are illustrated by an analysis of standard DNA fingerprints of Mycobacterium tuberculosis in San Francisco collected during the period 1991-1997. Graphical representation of genetic distances can assist in determining if the disappearance of a specific pattern in a community is due to interruption of transmission or ongoing evolution of the microorganism's fingerprint. Genetic distances can also compensate for varying information content derived by DNA fingerprints of contrasting pattern complexity. To study demographic and clinical correlates of transmission, the authors calculated the smallest genetic distance from each patient sample to all other samples. With correlation of genetic distances and nearest genetic distances with previously understood notions of the epidemiology of M. tuberculosis in San Francisco, factors influencing transmission are investigated.

Sex differences in the epidemiology of tuberculosis in San Francisco.

SETTING: Worldwide differences in sex-specific tuberculosis case rates remain fundamentally unexplained. OBJECTIVE: To explore various factors that may explain sex differences in tuberculosis incidence rates for San Francisco from 1991-1996. DESIGN: A retrospective epidemiologic analysis of sex-specific tuberculosis incidence rates in San Francisco from 1991-1996. Stratified analyses were performed on age at diagnosis, racial/ethnic group, human immunodeficiency virus (HIV) status, and place of birth. Molecular fingerprinting with IS6110 data was used to study sex differences in the incidence of disease for recently transmitted and reactivated cases of tuberculosis. RESULTS: In the study period, the male to female incidence rate ratio was 2.1 (95% CI 1.9-2.3). Stratified analyses revealed differences in sex-specific rates after the age of 14 and the highest male:female ratios were seen in the US-born, white, and black populations. High ratios were also observed for cases with clustered fingerprints, similar to those observed for the US-born population. In sub-populations with predominantly reactivated cases of tuberculosis, ratios were also above unity after adolescence, but the effect was less pronounced. CONCLUSION: The ongoing transmission of tuberculosis in the US-born population is one of the factors that explains the difference in sex-specific rates of disease in San Francisco. Observed differences in tuberculosis rates between the sexes may be due to a difference in transmission dynamics rather than diagnosis or reporting biases.