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RATIONALE AND OBJECTIVES: Identifying and treating persons with latent tuberculosis (TB) infection (LTBI) at high risk for developing TB is part of the current TB elimination strategy. There are no specific criteria, other than medical risks, to designate groups as high risk for developing TB. We hypothesized that, if location-based screenings were done in communities where persons with genotypically clustered Mycobacterium tuberculosis resided, then persons with LTBI from recent transmission and with undiagnosed TB could be identified. METHODS: Location-based TB screenings were done in partnership with multiple community-based organizations using resources previously used for other types of screening. MAIN RESULTS: Location-based screenings identified one person with TB for every 83 screened, and one person with LTBI for every five screened. The yield of this targeted screening program for discovering persons with TB and LTBI exceeded what would be expected from nontargeted screening in a county with a TB incidence of 5.7 per 100,000 population. CONCLUSIONS: Genotyping combined with geographic information systems analysis can potentially be used to define high-risk status and to define areas for location-based TB screenings. [ABSTRACT FROM AUTHOR]

This research follows the Updated Guidelines for Evaluating Public Health Surveillance Systems, Recommendations from the Guidelines Working Group, published by the Centers for Disease Control and Prevention nearly a decade ago. Since then, models have been developed and complex systems have evolved with a breadth of disparate data to detect or forecast chemical, biological, and radiological events that have a significant impact on the One Health landscape. How the attributes identified in 2001 relate to the new range of event-based biosurveillance technologies is unclear. This article frames the continuum of event-based biosurveillance systems (that fuse media reports from the internet), models (ie, computational that forecast disease occurrence), and constructs (ie, descriptive analytical reports) through an operational lens (ie, aspects and attributes associated with operational considerations in the development, testing, and validation of the event-based biosurveillance methods and models and their use in an operational environment). A workshop was held in 2010 to scientifically identify, develop, and vet a set of attributes for event-based biosurveillance. Subject matter experts were invited from 7 federal government agencies and 6 different academic institutions pursuing research in biosurveillance event detection. We describe 8 attribute families for the characterization of event-based biosurveillance: event, readiness, operational aspects, geographic coverage, population coverage, input data, output, and cost. Ultimately, the analyses provide a framework from which the broad scope, complexity, and relevant issues germane to event-based biosurveillance useful in an operational environment can be characterized.

Background: Directly observed therapy (DOT) is a widely recommended and promoted strategy to manage tuberculosis (TB), however, there is still disagreement about the role of DOT in TB control and the impact it has on reducing the acquisition and transmission of drug resistant TB. This study compares the portion of drug resistant genotype clusters, representing recent transmission, within and between communities implementing programs differing only in their directly observed therapy (DOT) practices., Methods: Genotype clusters were defined as 2 or more patient members with matching IS6110 restriction fragment length polymorphism (RFLP) and spoligotype patterns from all culture-positive tuberculosis cases diagnosed between January 1, 1995 and December 31, 2001. Logistic regression was used to compute maximum-likelihood estimates of odds ratios (ORs) and 95% confidence intervals (CIs) comparing cluster members with and without drug resistant isolates. In the universal DOT county, all patients received doses under direct observation of health department staff; whereas in selective DOT county, the majority of received patients doses under direct observation of health department staff, while some were able to self-administer doses., Results: Isolates from 1,706 persons collected during 1,721 episodes of tuberculosis were genotyped. Cluster members from the selective DOT county were more than twice as likely than cluster members from the universal DOT county to have at least one isolate resistant to isoniazid, rifampin, and/or ethambutol (OR = 2.3, 95% CI: 1.7, 3.1). Selective DOT county isolates were nearly 5 times more likely than universal DOT county isolates to belong to clusters with at least 2 resistant isolates having identical resistance patterns (OR = 4.7, 95% CI: 2.9, 7.6)., Conclusions: Universal DOT for tuberculosis is associated with a decrease in the acquisition and transmission of resistant tuberculosis.

Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. Rifampicin (RIF) resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE primer sets that scanned for DNA alterations across 775 bp of rpoB were developed. These primer sets were used to scan rpoB for DNA alterations in 296 M. tuberculosis patient isolates from the United States-Mexico border states of Texas and Tamaulipas. The most useful primer set scanned for mutations in the rifampin resistance-determining region (RRDR) and detected mutations in 95% of the RIF-resistant isolates compared to 2% of RIF-susceptible isolates. Thirty-four different alterations were observed within the RRDR by DGGE. In addition, isolates harboring mixtures of DNA within rpoB were readily detected by DGGE. A second PCR primer set was used to detect the V146A mutation in 5 to 7% of RIF-resistant isolates. A third primer set was used to detect mutations in 3% of RIF-resistant isolates, some of which also harbored mutations in the RRDR. Only 1 of 153 RIF-resistant isolates did not have a detectable rpoB mutation as determined by DGGE and DNA sequencing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with drug resistance in M. tuberculosis.

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90 degrees C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis.

BACKGROUND: Currently in the U.S. it is recommended that tuberculosis screening and treatment programs be targeted at high-risk populations. While a strategy of targeted testing and treatment of persons most likely to develop tuberculosis is attractive, it is uncertain how best to accomplish this goal. In this study we seek to identify geographical areas where on-going tuberculosis transmission is occurring by linking Geographic Information Systems (GIS) technology with molecular surveillance. METHODS: This cross-sectional analysis was performed on data collected on persons newly diagnosed with culture positive tuberculosis at the Tarrant County Health Department (TCHD) between January 1, 1993 and December 31, 2000. Clinical isolates were molecularly characterized using IS6110-based RFLP analysis and spoligotyping methods to identify patients infected with the same strain. Residential addresses at the time of diagnosis of tuberculosis were geocoded and mapped according to strain characterization. Generalized estimating equations (GEE) analysis models were used to identify risk factors involved in clustering. RESULTS: Evaluation of the spatial distribution of cases within zip-code boundaries identified distinct areas of geographical distribution of same strain disease. We identified these geographical areas as having increased likelihood of on-going transmission. Based on this evidence we plan to perform geographically based screening and treatment programs. CONCLUSION: Using GIS analysis combined with molecular epidemiological surveillance may be an effective method for identifying instances of local transmission. These methods can be used to enhance targeted screening and control efforts, with the goal of interruption of disease transmission and ultimately incidence reduction.

Molecular epidemiological tools for genotyping clinical isolates of Mycobacterium tuberculosis have been developed and used to help track and contain transmission of tuberculosis. We identified 87 short sequence repeat loci within the genome of the M. tuberculosis H37Rv strain. Nine tandem repeats were found to be variable (variable-number tandem repeats [VNTRs]) in a set of 91 isolates. Fifty-seven of the isolates had only four IS6110 bands. The other 34 isolates were members of the Beijing strain family. The number of alleles of each these nine VNTRs was determined by examining each isolate. Six of the loci (Mtb-v1, -v4, -v10, -v15, -v18, and -v20) were able to differentiate the Beijing spoligotype identical isolates into seven distinct genotypes. Five of the loci (Mtb-v3, -v5, -v6, -v10, and -v15) were informative in discriminating the four-band IS6110 restriction fragment length polymorphism isolates from each other. The Nei's diversity values of each marker ranged from 0.02 to 0.59, with the number of alleles ranging from two to eight across the entire strain set. These nine loci provide a useful, discriminatory extension of VNTR typing methods for application to molecular epidemiologic studies of M. tuberculosis.

To examine the transmission of drug-resistant (DR) tuberculosis between Texas and Mexico, Mycobacterium tuberculosis isolates resistant to one or more of the first-line antimycobacterial drugs were obtained from 606 patients who resided in Texas and 313 patients who resided in Mexico, primarily within the state of Tamaulipas. The isolates were genotyped by IS6110-based restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Of the 919 isolates genotyped, 413 (45%) grouped into 105 clusters containing 2 or more isolates with identical genotypes. In addition to having identical genotypes, identical drug resistance patterns were identified in 250 isolates in 78 clusters (DR clusters). Twenty DR clusters, containing isolates from 32% of the total number of patients infected with DR strains, were geographically distributed across Mexico and Texas. Within this population of 919 patients infected with DR isolates, the probability of being in a DR cluster was the same for residents of Mexico and Texas. In Texas, the significant independent predictors of clustering within DR clusters as opposed to genotype clusters were found to be race, age, country of birth, human immunodeficiency virus (HIV) infection status, and resistance to more than one drug. Specifically, isolates from African Americans, individuals under age 65, individuals born in the United States, and HIV-positive individuals were each more likely to be associated with a DR cluster. By contrast, no significant independent predictors of clustering in a DR cluster were identified in Mexico. Although some DR M. tuberculosis strains are geographically restricted, this study suggests that a number of strains are transmitted between Mexico and the United States.

Previous studies from our laboratory established that C-ASWS, an alkali-soluble, water-soluble extract from cell walls of Coccidioides immitis, protects mice against lethal challenge with this fungus. The C-ASWS extract contains a glycosylated protein, designated antigen 2 (Ag2), and a polysaccharide antigen. We recently cloned Ag2 cDNA and showed that the recombinant fusion protein elicited strong delayed-type hypersensitivity responses in immunized mice. This investigation was undertaken to determine if the recombinant Ag2 protein, expressed as an Ag2-glutathione S-transferase (GST) fusion protein, or Ag2 cDNA would protect mice against lethal challenge with C. immitis. The recombinant Ag2-GST protein protected BALB/c mice against intraperitoneal challenge with 250 arthroconidia, as assessed by a decrease in fungal CFU in tissues. The Ag2-GST-immunized mice did not show, however, an increased survival during a 30-day period postinfection. By contrast, immunization of mice with Ag2 cDNA ligated into the pVR1012 plasmid engendered protection against intraperitoneal challenge with 2,500 arthroconidia and against pulmonary challenge with 50 arthroconidia. Vaccine efficacy paralleled the development of delayed-type hypersensitivity responses to C. immitis antigen. Whereas mice vaccinated with the recombinant Ag2-GST protein did not mount footpad hypersensitivity to C-ASWS or the recombinant Ag2-GST protein, mice vaccinated with the pVR1012-Ag2 construct mounted a strong footpad hypersensitivity and their spleen cells secreted gamma interferon upon in vitro stimulation with the Ag2-containing C-ASWS extract. This is the first investigation to show that genetic immunization can protect against lethal challenge with C. immitis.