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Background Recognizing pulmonary involvement in tuberculosis (TB) patients is necessary to prevent TB transmission. We describe frequency and characteristics of patients with extrapulmonary TB (EPTB), normal chest radiographs, and positive sputum culture. Methods We analyzed data of patients ≥15 years of age with EPTB reported to the California TB registry during 2011–2017 with cultured sputum and normal chest radiographs using generalized linear modeling to estimate prevalence ratios associated with positive sputum culture. Demographic, behavioral, clinical characteristics, and testing were compared for patients with positive and negative sputum culture. Results Of 1635 patients with EPTB and normal chest radiographs, 937 (57%) had sputum culture performed, and 127 (13%) patients had positive results for Mycobacterium tuberculosis complex. Patients with positive results were more likely to: be male, experience homelessness, use substances, have HIV, and have >1 disease site. Among 85 patients with HIV co-infection, 54% had positive culture results compared with 9.5% among 852 patients without HIV co-infection. Patients with EPTB in more than 1 site were also more likely to have a positive sputum culture. Conclusions Culturing sputum from patients with EPTB identified pulmonary cases not detected by chest radiograph, particularly among patients with HIV or >1 disease site.

BACKGROUND: Treatment of latent tuberculosis infection is an important strategy to prevent tuberculosis disease. In the USA, three tests are used to identify latent tuberculosis infection: the tuberculin skin test (TST) and two IFN-γ release assays (T-SPOT.TB and QuantiFERON). To our knowledge, few large studies have compared all three tests among people at high risk of latent tuberculosis infection or progression to tuberculosis disease. We aimed to assess test agreement between IFN-γ release assays and TST to provide guidance on their use in important risk groups. METHODS: In this observational cohort study, we enrolled participants at high risk of latent tuberculosis infection or progression to tuberculosis disease at ten US sites with 18 affiliated clinics, including close contacts of infectious tuberculosis cases, people born in countries whose populations in the USA have high (≥100 cases per 100 000 people) or moderate (10–99 cases per 100 000 people) tuberculosis incidence, and people with HIV. Participants were interviewed about demographics and medical risk factors, and all three tests were administered to each participant. The primary endpoints for this study were the proportions of positive test results by test type stratified by risk group and test concordance by risk group for participants with valid results for all three test types. The study is registered at ClinicalTrials.gov, NCT01622140. FINDINGS: Between July 12, 2012, and May 5, 2017, 26 292 people were approached and 22 131 (84·2%) were enrolled in the study. Data from 21 846 (98·7%) participants were available for analysis, including 3790 (17·3%) born in the USA and 18 023 (82·5%) born outside the USA. Among non-US-born participants overall, the RR comparing the proportions of TST-positive results (7476 [43·2%] of 17 306 participants) to QuantiFERON-positive results (4732 [26·5%] of 17 882 participants) was 1·6 (95% CI 1·6–1·7). The risk ratio (RR) for the comparison with the proportion of T-SPOT.TB-positive results (3693 [21·6%] of 17 118 participants) was 2·0 (95% CI 1·9–2·1). US-born participants had less variation in the proportions of positive results across all tests. The RRs for the proportion of TST-positive results (391 [10·9%] of 3575 participants) compared with the proportion of QuantiFERON-positive results (445 [12·0%] of 3693 participants) and T-SPOT.TB-positive results (295 [8·1%] of 3638 participants) were 0·9 (95% CI 0·8–1·0) and 1·3 (1·2–1·6), respectively. 20 149 (91·0%) of 21 846 participants had results for all three tests, including 16 712 (76%) non-US-born participants. Discordance between TST and IFN-γ release assay results varied by age among non-US-born participants and was greatest among the 848 non-US-born children younger than 5 years. 204 (87·2%) of 234 non-US-born children younger than 5 years with at least one positive test were TST-positive and IFN-γ release assay-negative. The proportion of non-US-born participants who were TST-negative but IFN-γ release assay-positive ranged from one (0·5%) of 199 children younger than 2 years to 86 (14·5%) of 594 participants aged 65 years and older (p(trend)

Trees can sequester air pollutants, and air pollution is associated with poor tuberculosis outcomes. However, the health impacts of urban trees on tuberculosis patients are unknown. To elucidate the effects of urban tree canopy on mortality during tuberculosis treatment, we evaluated patients diagnosed with active tuberculosis in California from 2000 through 2012, obtaining patient data from the California tuberculosis registry. Our primary outcome was all-cause mortality during tuberculosis treatment. We determined percent tree cover using 1mresolution color infrared orthoimagery categorized into land cover classes, then linked tree cover to four circular buffer zones of 50-300m radii around patient residential addresses. We used the Kaplan-Meier method to estimate survival probabilities and Cox regression models to determine mortality hazard ratios, adjusting for demographic, socioeconomic, and clinical covariates. Our cohort included 33,962 tuberculosis patients of median age 47, 59% male, 51% unemployed, and 4.9% HIV positive. Tuberculosis was microbiologically confirmed in 79%, and 1.17% were multi-drug resistant (MDR). Median tree cover was 7.9% (50m buffer). Patients were followed for 23,280 person-years with 2370 deaths during tuberculosis treatment resulting in a crude mortality rate of 1018 deaths per 10,000 person-years. Increasing tree cover quintiles were associated with decreasing mortality risk during tuberculosis treatment in all buffers, and the magnitude of association decreased incrementally with increasing buffer radius: In the 50m buffer, patients living in neighborhoods with the highest quintile tree cover experienced a 22% reduction in mortality (HR 0.78, 95%CI 0.68-0.90) compared to those living in lowest quintile tree cover; whereas for 100, 200, and 300m buffers, a 21%, 13%, and 11% mortality risk reduction was evident. In conclusion, urban tree canopy was associated with decreased mortality during tuberculosis treatment even after adjusting for multiple demographic, socioeconomic, and clinical factors, suggesting that trees might play a role in improving tuberculosis outcomes.

Setting California, United States. Objective To determine the frequency of tuberculosis (TB) patients at risk for relapse who received at least 9 months of anti-tuberculosis treatment (extended treatment) and to identify factors associated with not receiving extended treatment. Design We analyzed characteristics of culture-confirmed pulmonary TB patients reported to the California TB Registry during 2004-2009. Patients with cavities on initial chest radiograph and delayed culture conversion (⩾70 days) were at 'high risk of relapse', and anti-tuberculosis treatment of ⩾270 days was 'extended treatment'. We used a generalized linear model to identify independent risk factors for absence of extended treatment in the high risk of relapse group. Results Among 5680 TB patients, 483 (8.5%) were at high risk of relapse: 372 (77%) received extended treatment but 111 (23%) did not. Factors associated with absence of extended treatment included negative sputum smears (adjusted prevalence ratio [aPR] 2.62, 95%CI 1.69-4.05), residence in three specific counties (aPR 1.71, 95%CI 1.19-2.46) and Black race (aPR 1.56, 95%CI 1.03-2.38). Conclusions Nearly a quarter of TB patients at high risk of relapse did not receive extended treatment. Increased efforts are needed to ensure that all patients who may benefit from extended anti-tuberculosis treatment receive it.

Setting Studies of US populations have produced conflicting findings about the impact of diabetes mellitus (DM) on tuberculosis (TB) treatment outcomes. Objective To investigate the association between DM and all-cause mortality among patients on anti-tuberculosis treatment in California, USA. Design Using TB surveillance data, we conducted a retrospective analysis of California patients with culture-confirmed TB who started anti-tuberculosis treatment during 2010-2014. We used Cox proportional hazards models to estimate the association of DM with all-cause mortality and conducted a sensitivity analysis to estimate the attenuating effect of unmeasured confounding by body mass index. Results Among 8461 patients with TB, 2124 (25.1%) had DM and 713 (8.4%) died during anti-tuberculosis treatment. A higher proportion of TB-DM patients died (13.1% vs. 6.8% TB-no DM). After adjusting for confounders, DM was associated with mortality (adjusted hazards ratio [aHR] 1.35, 95%CI 1.15-1.57). There was effect modification by human immunodeficiency virus (HIV) status, with HIV-positive patients having an aHR of 5.33 (95%CI 1.76-16.12). Conclusion TB patients with DM had a greater hazard of death during anti-tuberculosis treatment than those without DM. Further investigation into the impact of HIV on the relation of DM to death is necessary.

Rationale: More information on risk factors for death from tuberculosis in the United States could help reduce the tuberculosis mortality rate, which has remained steady for more than a decade.Objective: To identify risk factors for tuberculosis-related death in adults.Methods: We performed a retrospective study of 1,304 adults with tuberculosis who died before treatment completion and 1,039 frequency-matched control subjects who completed tuberculosis treatment in 2005 to 2006 in 13 states reporting 65% of U.S. tuberculosis cases. We used in-depth record abstractions and a standard algorithm to classify deaths in persons with tuberculosis as tuberculosis-related or not. We then compared these classifications to causes of death as coded in death certificates. We used multivariable logistic regression to calculate adjusted odds ratios for predictors of tuberculosis-related death among adults compared with those who completed tuberculosis treatment.Results: Of 1,304 adult deaths, 942 (72%) were tuberculosis related, 272 (21%) were not, and 90 (7%) could not be classified. Of 847 tuberculosis-related deaths with death certificates available, 378 (45%) did not list tuberculosis as a cause of death. Adjusting for known risks, we identified new risks for tuberculosis-related death during treatment: absence of pyrazinamide in the initial regimen (adjusted odds ratio, 3.4; 95% confidence interval, 1.9-6.0); immunosuppressive medications (adjusted odds ratio, 2.5; 95% confidence interval, 1.1-5.6); incomplete tuberculosis diagnostic evaluation (adjusted odds ratio, 2.2; 95% confidence interval, 1.5-3.3), and an alternative nontuberculosis diagnosis before tuberculosis diagnosis (adjusted odds ratio, 1.6; 95% confidence interval, 1.2-2.2).Conclusions: Most persons who died with tuberculosis had a tuberculosis-related death. Intensive record review revealed tuberculosis as a cause of death more often than did death certificate diagnoses. New tools, such as a tuberculosis mortality risk score based on our study findings, may identify patients with tuberculosis for in-hospital interventions to prevent death.

Background: Ambient air pollution and tuberculosis (TB) have an impact on public health worldwide, yet associations between the two remain uncertain. Objective: We determined the impact of resident...

Introduction: Trees can mitigate air pollution, but health benefits remain unclear. Methods: We evaluated the effects of urban tree coverage on mortality in a large cohort of Californians with acti...

Since 1992, Mycobacterium tuberculosis strain PG004 has been responsible for a large outbreak of tuberculosis in one northern Californian community. There are no epidemiological or host factors to explain this outbreak. PG004 was therefore analysed for biological characteristics that might explain its widespread distribution. BABL/c mice were infected intravenously with PG004, non-PG004 M. tuberculosis strains CCC20 and CCC23 isolated from patients in the same community, and the laboratory strain H37Rv. The susceptibility of PG004 to reactive nitrogen intermediates (RNIs) was compared with that of H37Rv. Because of the reported association of phenolic glycolipid production with mouse virulence, a junction sequence in the polyketide synthase gene cluster (pks 15/1) was compared among strains. It was found that the most virulent strain, based on mouse mortality, was not the outbreak strain PG004, but the non-outbreak strain CCC20. This strain had an intact pks 15/1 sequence identical to that of another non-outbreak strain, CCC23, which caused death in only one out of ten mice in 300 days of follow-up. The outbreak strain PG004 had a frameshift mutation in the pks 15/1 sequence identical to the sequence of H37Rv, and it was no more resistant to RNIs than H37Rv. The most distinguishing feature of PG004 was its failure to produce well-organized, coalescing granulomas in mouse lungs. The lack of organized granulomas and reduced pathology may prevent restriction of PG004 in the lungs and allow it to spread into alveolar air spaces and escape the host to transmit to others. Humans with reduced lung pathology may remain undiagnosed and untreated in the community longer than those with severe disease. The over-representation of an M. tuberculosis strain in a community, therefore, may be more associated with strains that cause reduced rather than severe lung pathology.

Background Nucleic acid amplification tests (NAATs) have been used as a diagnostic tool for tuberculosis (TB) in the United States for many years. We sought to assess NAAT use in TB patients in California during a period of time when NAAT availability increased throughout the world. Methods We conducted a retrospective review of surveillance data from 6051 patients with culture-confirmed pulmonary TB who were reported to the California TB registry during 2010–2013. Results Only 2336 of 6051 (39%) TB patients had a NAAT for diagnosis before culture results. Although 90% (N = 2101) with NAAT had positive test results, 9% (N = 217) had falsely negative NAAT results, and 0.8% (N = 18) had indeterminate NAAT results. The median time from specimen collection to TB treatment initiation was shorter when NAAT was used (3 vs 14 days, P < .0001), and patients with a positive NAAT result initiated treatment earlier than patients with a falsely negative result (1 vs 11 days from NAAT report, P < .0001). We confirmed the increased sensitivity of NAAT compared with acid-fast bacilli (AFB) smear microscopy in our study population; 92 of 145 AFB smear-negative patients had positive NAATs. Median time from specimen collection to NAAT result report differed by health jurisdiction, from 1 to 11 working days. Conclusions Increased use of NAATs in diagnosis of pulmonary TB could decrease the time-to-treatment initiation and consequently decrease transmission. However, differential use and access to NAAT may prevent full realization of NAAT benefits in California.

To summarize, using meta-analysis, the accuracy of bacteriophage-based assays for the detection of rifampicin resistance in Mycobacterium tuberculosis.By searching multiple databases and sources we identified a total of 21 studies eligible for meta-analysis. Of these, 14 studies used phage amplification assays (including eight studies on the commercial FASTPlaque-TB kits), and seven used luciferase reporter phage (LRP) assays. Sensitivity, specificity, and agreement between phage assay and reference standard (e.g. agar proportion method or BACTEC 460) results were the main outcomes of interest.When performed on culture isolates (N=19 studies), phage assays appear to have relatively high sensitivity and specificity. Eleven of 19 (58%) studies reported sensitivity and specificity estimatesor =95%, and 13 of 19 (68%) studies reportedor =95% agreement with reference standard results. Specificity estimates were slightly lower and more variable than sensitivity; 5 of 19 (26%) studies reported specificity90%. Only two studies performed phage assays directly on sputum specimens; although one study reported sensitivity and specificity of 100 and 99%, respectively, another reported sensitivity of 86% and specificity of 73%.Current evidence is largely restricted to the use of phage assays for the detection of rifampicin resistance in culture isolates. When used on culture isolates, these assays appear to have high sensitivity, but variable and slightly lower specificity. In contrast, evidence is lacking on the accuracy of these assays when they are directly applied to sputum specimens. If phage-based assays can be directly used on clinical specimens and if they are shown to have high accuracy, they have the potential to improve the diagnosis of MDR-TB. However, before phage assays can be successfully used in routine practice, several concerns have to be addressed, including unexplained false positives in some studies, potential for contamination and indeterminate results.

Prompt laboratory reporting of tuberculosis (TB) test results is necessary for TB control. To understand the extent of and factors contributing to laboratory reporting delays and the impact of reporting delays on initiation of treatment of TB patients, we analyzed data from 300 consecutive culture-positive TB cases reported in four California counties in 1998. Laboratory reporting to the specimen submitter was delayed for 26.9% of smear-positive patients and 46.8% of smear-negative patients. Delays were associated with the type of laboratory that performed the testing and with delayed transport of specimens. Referral laboratories (public health and commercial) had longer median reporting time frames than hospital and health maintenance organization laboratories. Among patients whose treatment was not started until specimens were collected, those with delayed laboratory reporting were more likely to have delayed treatment than patients with no laboratory reporting delays (odds ratio [OR] of 3.9 and 95% confidence interval [CI] of 1.6 to 9.7 for smear-positive patients and OR of 13.1 and CI of 5.3 to 32.2 for smear-negative patients). This relation remained after adjustment in a multivariate model for other factors associated with treatment delays (adjusted OR of 25.64 and CI of 7.81 to 83.33 for smear-negative patients). These findings emphasize the need to reduce times of specimen transfer between institutions and to ensure rapid communication among laboratories, health care providers, and health departments serving TB patients.

Background. Identifying factors associated with tuberculosis (TB) deaths will inform efforts to prevent deaths. Methods. We examined deaths among patients with culture-confirmed TB reported to the California TB Registry during 1994–2008. We calculated the age-adjusted percentage of deaths before and during TB treatment and estimated trends. We constructed multivariable logistic regression models to identify factors associated with death during treatment. Results. Of 40 125 patients with culture-confirmed TB, 4565 (11%) died: 1146 (25%) died before treatment started, and 3419 (75%) died during treatment. The age-adjusted percentage of patients who died before and during treatment declined from 1994 to 2008 (3.5% to 2%, and 10.4% to 7.2%, respectively, both P < .0001). We identified several risk factors for death that may be addressed with public health efforts: acquired multidrug resistance (adjusted odds ratio [aOR] = 4.67; 95% confidence interval [CI], 2.09–10.45); care in the private sector (aOR = 3.08; 95% CI, 2.75–3.44); and an initial treatment regimen of Conclusions. In California, 1 in 9 TB patients died with a potentially curable disease. Public health departments might prevent deaths in patients with TB by strengthening partnerships with private providers, intensifying diagnostic and treatment processes for patients at risk of death in hospital, optimizing treatment regimens for patients with comorbidities, and preventing the acquisition of drug resistance.

Summary Diaminopimelic acid (DAP) is a major component of the peptidoglycan layer of the mycobacterial cell wall. The mycobacterial cell wall has been implicated as a potential virulence factor and is highly immunogenic. The pathway for biosynthesis of DAP may serve as a target in the design of antimycobacterial agents and construction of in vivo selection systems. Despite its significance, this biosynthetic pathway is poorly understood in mycobacteria. In order to develop a better understanding of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehydrogenase (asd) genes of Mycobacterium smegmatis, bacille Calmette-Guerin (BCG), Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis were isolated. The M. smegmatis asd gene was isolated by complementation in Escherichia coli. This gene was then used to isolate the asd genes from other mycobacteria. The asd-complemening fragments from BCG and M. smegmatis were sequenced. An open reading frame upstream of the mycobacterial asd gene was identified as the mycobacterial aspartokinase gene (ask). Primer extension analysis revealed that the only transcriptional start in this region is found 5’of the ask gene. This observation indicates that the mycobacterial ask and asd genes are in an operon.

Background Recurrent tuberculosis suggests potentially modifiable gaps in tuberculosis treatment and control activities. The frequency of late recurrences following treatment completion has not been well-studied. We determined the frequency of, and risk factors associated with, tuberculosis that recurs at least one year after completion of anti-tuberculosis therapy in California. Methods The study population included culture-positive, pulmonary tuberculosis patients reported to the California tuberculosis case registry from 1993 to 2007 who completed anti-tuberculosis therapy. A person with late recurrent tuberculosis was defined as an individual that appeared in the registry more than once, determined by match on name and date-of-birth, with at least one year between treatment completion of the first episode and treatment initiation of the second episode. Results Among 23,517 tuberculosis patients, 148 (0.63%) had a late recurrence. Independent risk factors for recurrence included: infection with a pyrazinamide mono-resistant isolate (adjusted hazard ratio, 2.93; p = 0.019); initiation of an isoniazid- and rifampin-only treatment regimen (adjusted hazard ratio, 2.55; p = 0.0412); sputum smear-positive disease (adjusted hazard ratio, 1.96; p = 0.0003); human immunodeficiency virus infection (adjusted hazard ratio, 1.81; p = 0.0149); and birth in the United States (adjusted hazard ratio, 1.88; p = 0.0002). Infection with an isoniazid mono-resistant isolate was protective (adjusted hazard ratio, 0.25; p = 0.0171). Conclusions The low frequency of late recurrent tuberculosis in California suggests that local TB control programs are largely successful at preventing this adverse outcome. Nonetheless, we identified subpopulations at increased risk of late tuberculosis recurrence that may benefit from additional medical or public health interventions.

We have developed a novel method for screening a Mycobacterium bovis (BCG) cosmid library in Mycobacterium smegmatis for the detection of immunostimulatory T-cell antigens (Ag). Distinctive protein banding patterns were demonstrated in culture filtrates of three of 30 recombinant M. smegmatis clones: pBCCS13 (41 and 73 kDa); pBCCS221 (30, 50 and 68 kDa); pBCCS223 (100 kDa). Western immunoblots indicated that monoclonal antibodies (mAb) directed to the previously characterized 19-, 30-, 38-, 65- and 71-kDa mycobacterial Ag were not reactive with the distinctive recombinant proteins. Furthermore, T-cell Western blots demonstrated that fractions containing the distinctive proteins were immunostimulatory. A given tuberculin-positive donor expressed unique patterns of blastogenic reactivity to protein fractions isolated from each of the three recombinant clones. Restriction enzyme digests of the three recombinant BCG inserts revealed distinctive DNA-banding patterns. The immunostimulatory Ag, therefore, are most likely encoded within different regions of the BCG genome, as contained within three distinct inserts. T-cell Western blots further indicated a heterogeneity in the repertoire of BCG-responsive T cells since tuberculin-positive donors varied in the pattern of reactivity to protein fractions isolated from the same recombinant filtrate. Most likely, immunity to M. tuberculosis results from activation of a heterogeneous array of T cells targeted to multiple immunostimulatory Ag. The method we describe should greatly enhance our ability to define the full spectrum of T-cell Ag encoded by mycobacteria, particularly those which are secreted proteins.

Background Sputum microscopy, the most important conventional test for tuberculosis, is specific in settings with high burden of tuberculosis and low prevalence of non tuberculous mycobacteria. However, the test lacks sensitivity. Although bacteriophage-based tests for tuberculosis have shown promising results, their overall accuracy has not been systematically evaluated. Methods We did a systematic review and meta-analysis of published studies to evaluate the accuracy of phage-based tests for the direct detection of M. tuberculosis in clinical specimens. To identify studies, we searched Medline, EMBASE, Web of science and BIOSIS, and contacted authors, experts and test manufacturers. Thirteen studies, all based on phage amplification method, met our inclusion criteria. Overall accuracy was evaluated using forest plots, summary receiver operating (SROC) curves, and subgroup analyses. Results The data suggest that phage-based assays have high specificity (range 0.83 to 1.00), but modest and variable sensitivity (range 0.21 to 0.88). The sensitivity ranged between 0.29 and 0.87 among smear-positive, and 0.13 to 0.78 among smear-negative specimens. The specificity ranged between 0.60 and 0.88 among smear-positive and 0.89 to 0.99 among smear-negative specimens. SROC analyses suggest that overall accuracy of phage-based assays is slightly higher than smear microscopy in direct head-to-head comparisons. Conclusion Phage-based assays have high specificity but lower and variable sensitivity. Their performance characteristics are similar to sputum microscopy. Phage assays cannot replace conventional diagnostic tests such as microscopy and culture at this time. Further research is required to identify methods that can enhance the sensitivity of phage-based assays without compromising the high specificity.

Mycobacterium ulceransis the causative agent of Buruli ulcer, a tropical ulcerative skin disease. One of the most intriguing aspects of this disease is the presence of extensive tissue damage in the absence of an acute inflammatory response. We recently purified and characterized a macrolide toxin, mycolactone, fromM. ulcerans. Injection of this molecule into guinea pig skin reproduced cell death and lack of acute inflammatory response similar to that seen following the injection of viable bacteria. We also showed that mycolactone causes a cytopathic effect on mouse fibroblast L929 cells that is characterized by cytoskeletal rearrangements and growth arrest within 48 h. However, these results could not account for the extensive cell death which occurs in Buruli ulcer. The results presented here demonstrate that L929 and J774 mouse macrophage cells die via apoptosis after 3 to 5 days of exposure to mycolactone. Treatment of cells with a pan-caspase inhibitor can inhibit mycolactone-induced apoptosis. We demonstrate that injection of mycolactone into guinea pig skin results in cell death via apoptosis and that the extent of apoptosis increases as the lesion progresses. These results may help to explain why tissue damage in Buruli ulcer is not accompanied by an acute inflammatory response.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.

Salmonella typhi and Salmonella gallinarum phenotypes correlated with mouse host restriction have been identified by using in vitro and in vivo systems. S. typhi is capable of entering the murine intestinal epithelium via M cells, as is Salmonella typhimurium, which causes systemic infection in the mouse. But, unlike S. typhimurium, S. typhi does not destroy the epithelium and is cleared from the Peyer's patches soon after M-cell entry. S. gallinarum appears to be incapable of entering the murine Peyer's patch epithelium. Our in vitro evidence suggests that S. gallinarum is taken up in murine phagocytic cells by a mechanism different from that of S. typhimurium. S. typhimurium is taken up at a higher frequency and is maintained at higher viable counts throughout a 24-h time course in a murine macrophage-like cell line than are S. gallinarum and S. typhi.

Enteric microbial pathogens interact with the gut epithelium to establish infection. Recently, it has become clear that many microorganisms that colonize or traverse the intestinal mucosa do so via the specialized M cells. Recent work has shown that Shigella flexneri and Salmonella typhimurium specifically target M cells to initiate infection of the host.

Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.

Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the Mycobacterium smegmatis chromosome. We have identified the int gene and attP site of L5, characterized the chromosomal attachment site (attB), and constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. These integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the pathogen Mycobacterium tuberculosis and the vaccine strain bacille Calmette-Guérin (BCG). The ability to easily generate stable recombinants in these slow-growing mycobacteria without the requirement for continual selection is of particular importance for the construction of recombinant BCG vaccines and for the isolation and characterization of mycobacterial pathogenic determinants in animal model systems. Integration vectors of this type should be of general use in a number of additional bacterial systems where temperate phages have been identified.

Publisher Summary This chapter discusses genetic systems for mycobacteria. The ability to perform genetic analyses on bacteria has provided powerful tools and experimental systems to unravel fundamental biological processes. The advances of recombinant DNA technologies have ignited the development of genetic systems for bacteria that are difficult to work with. The genus Mycobacterium contains a set of the most difficult bacterial species to manipulate experimentally. The tuberculosis vaccine strain, bacille Calmette Guerin (BCG) has been used to vaccinate more individuals than any other live bacterial vaccine, yet little is known about mycobacterial gene structure and expression. The recent development of phage, plasmid, and gene replacement systems for the introduction of recombinant DNA into mycobacteria has opened up a new era of research on members of the genus Mycobacterium .

Background For a regional project in four low-incidence states, we designed a customizable tuberculosis outbreak response plan. Prior to dissemination of the plan, a tuberculosis outbreak occurred, presenting an opportunity to perform a field assessment of the plan. The purpose of the assessment was to ensure that the plan included essential elements to help public health professionals recognize and respond to outbreaks. Methods We designed a semi-structured questionnaire and interviewed all key stakeholders involved in the response. We used common themes to assess validity of and identify gaps in the plan. A subset of participants provided structured feedback on the plan. Results We interviewed 11 public health and six community stakeholders. The assessment demonstrated that (1) almost all of the main response activities were reflected in the plan; (2) the plan added value by providing a definition of a tuberculosis outbreak and guidelines for communication and evaluation. These were areas that lacked written protocols during the actual outbreak response; and (3) basic education about tuberculosis and the interpretation and use of genotyping data were important needs. Stakeholders also suggested adding to the plan questions for evaluation and a section for specific steps to take when an outbreak is suspected. Conclusion An interactive field assessment of a programmatic tool revealed the value of a systematic outbreak response plan with a standard definition of a tuberculosis outbreak, guidelines for communication and evaluation, and response steps. The assessment highlighted the importance of education and training for tuberculosis in low-incidence areas.