Characterizing the etiology of recurrent tuberculosis using whole genome sequencing-Alaska, USA, 2008-2020.

BACKGROUND: Understanding the etiology of recurrent tuberculosis (rTB) is important for effective TB control. Prior to the advent of whole genome sequencing (WGS), attributing rTB to relapse or reinfection using genetic information was complicated by the limited resolution of conventional genotyping methods. METHODS: We applied a systematic method of evaluating whole genome single nucleotide polymorphism (wgSNP) distances and results of phylogenetic analyses to characterize the etiology of rTB in American Indian and Alaska Native (AIAN) persons in Alaska during 2008-2020. We contextualized our findings through descriptive analyses of surveillance data and results of a literature search for investigations that characterized rTB etiology using WGS. RESULTS: The percentage of TB cases in AIAN persons in Alaska classified as recurrent episodes (11.8%) was three times the national percentage (3.9%). Of 38 recurrent episodes included in genetic analyses, we attributed 25 (65.8%) to reinfection based on wgSNP distances and phylogenetic analyses; this proportion was the highest among 16 published point estimates identified through the literature search. By comparison, we attributed 11 of 38 (28.9%) and 6 of 38 (15.8%) recurrent episodes to reinfection based on wgSNP distances alone and on conventional genotyping methods, respectively. CONCLUSIONS: WGS and attribution criteria involving genetic distances and patterns of relatedness can provide an effective means of elucidating rTB etiology. Our findings indicate that rTB occurs at high proportions among AIAN persons in Alaska and is frequently attributable to reinfection, reinforcing the importance of active surveillance and control measures to limit the spread of TB disease in Alaskan AIAN communities.

Second Nationwide Tuberculosis Outbreak Caused by Bone Allografts Containing Live Cells - United States, 2023.

During July 7-11, 2023, CDC received reports of two patients in different states with a tuberculosis (TB) diagnosis following spinal surgical procedures that used bone allografts containing live cells from the same deceased donor. An outbreak associated with a similar product manufactured by the same tissue establishment (i.e., manufacturer) occurred in 2021. Because of concern that these cases represented a second outbreak, CDC and the Food and Drug Administration worked with the tissue establishment to determine that this product was obtained from a donor different from the one implicated in the 2021 outbreak and learned that the bone allograft product was distributed to 13 health care facilities in seven states. Notifications to all seven states occurred on July 12. As of December 20, 2023, five of 36 surgical bone allograft recipients received laboratory-confirmed TB disease diagnoses; two patients died of TB. Whole-genome sequencing demonstrated close genetic relatedness between positive Mycobacterium tuberculosis cultures from surgical recipients and unused product. Although the bone product had tested negative by nucleic acid amplification testing before distribution, M. tuberculosis culture of unused product was not performed until after the outbreak was recognized. The public health response prevented up to 53 additional surgical procedures using allografts from that donor; additional measures to protect patients from tissue-transmitted M. tuberculosis are urgently needed.

Broadband Data Forensics: Spatiotemporal Variations of the Download/Upload Speed Metric Commonly Used to Evaluate Potential Telehealth Accessibility.

Introduction: The complicated task of evaluating potential telehealth access begins with the metrics and supporting datasets that seek toevaluate the presence and durability of broadband connections in a community. Broadband download/upload speeds are one of the popular metrics used to measure potential telehealth access, which is critical to health equity. An understanding of the limitations of these measures is important for drawing conclusions about the reality of the digital divide in telehealth access. The objective of this study was to assess spatiotemporal variations in broadband download/upload speeds. Method: We analyzed a sample of data from the Speedtest Intelligence Portal provided through the Ookla for Good initiative. Results: We found that variation is inherent across the states of Vermont, New Hampshire, Louisiana, and Utah. Conclusions: The variation suggests that when single measures of download/upload speeds are used to evaluate telehealth accessibility they may be masking the true magnitude of the digital divide.

Molecular surveillance for large outbreaks of tuberculosis in the United States, 2014-2018.

OBJECTIVE: This study describes characteristics of large tuberculosis (TB) outbreaks in the United States detected using novel molecular surveillance methods during 2014-2016 and followed for 2 years through 2018. METHODS: We developed 4 genotype-based detection algorithms to identify large TB outbreaks of ≥10 cases related by recent transmission during a 3-year period. We used whole-genome sequencing and epidemiologic data to assess evidence of recent transmission among cases. RESULTS: There were 24 large outbreaks involving 518 cases; patients were primarily U.S.-born (85.1%) racial/ethnic minorities (84.1%). Compared with all other TB patients, patients associated with large outbreaks were more likely to report substance use, homelessness, and having been diagnosed while incarcerated. Most large outbreaks primarily occurred within residences among families and nonfamilial social contacts. A source case with a prolonged infectious period and difficulties in eliciting contacts were commonly reported contributors to transmission. CONCLUSION: Large outbreak surveillance can inform targeted interventions to decrease outbreak-associated TB morbidity.

Nationwide tuberculosis outbreak in the USA linked to a bone graft product: an outbreak report.

BACKGROUND: Mycobacterium tuberculosis transmission through solid organ transplantation has been well described, but transmission through transplanted tissues is rare. We investigated a tuberculosis outbreak in the USA linked to a bone graft product containing live cells derived from a single deceased donor. METHODS: In this outbreak report, we describe the management and severity of the outbreak and identify opportunities to improve tissue transplant safety in the USA. During early June, 2021, the US Centers for Disease Control and Prevention (CDC) worked with state and local health departments and health-care facilities to locate and sequester unused units from the recalled lot and notify, evaluate, and treat all identified product recipients. Investigators from CDC and the US Food and Drug Administration (FDA) reviewed donor screening and tissue processing. Unused product units from the recalled and other donor lots were tested for the presence of M tuberculosis using real-time PCR (rt PCR) assays and culture. M tuberculosis isolates from unused product and recipients were compared using phylogenetic analysis. FINDINGS: The tissue donor (a man aged 80 years) had unrecognised risk factors, symptoms, and signs consistent with tuberculosis. Bone was procured from the deceased donor and processed into 154 units of bone allograft product containing live cells, which were distributed to 37 hospitals and ambulatory surgical centres in 20 US states between March 1 and April 2, 2021. From March 3 to June 1, 2021, 136 (88%) units were implanted into 113 recipients aged 24-87 years in 18 states (some individuals received multiple units). The remaining 18 units (12%) were located and sequestered. 87 (77%) of 113 identified product recipients had microbiological or imaging evidence of tuberculosis disease. Eight product recipients died 8-99 days after product implantation (three deaths were attributed to tuberculosis after recognition of the outbreak). All 105 living recipients started treatment for tuberculosis disease at a median of 69 days (IQR 56-81) after product implantation. M tuberculosis was detected in all eight sequestered unused units tested from the recalled donor lot, but not in lots from other donors. M tuberculosis isolates from unused product and recipients were more than 99·99% genetically identical. INTERPRETATION: Donor-derived transmission of M tuberculosis via bone allograft resulted in substantial morbidity and mortality. All prospective tissue and organ donors should be routinely assessed for tuberculosis risk factors and clinical findings. When these are present, laboratory testing for M tuberculosis should be strongly considered. FUNDING: None.

Logically Inferred Tuberculosis Transmission (LITT): A Data Integration Algorithm to Rank Potential Source Cases.

Understanding tuberculosis (TB) transmission chains can help public health staff target their resources to prevent further transmission, but currently there are few tools to automate this process. We have developed the Logically Inferred Tuberculosis Transmission (LITT) algorithm to systematize the integration and analysis of whole-genome sequencing, clinical, and epidemiological data. Based on the work typically performed by hand during a cluster investigation, LITT identifies and ranks potential source cases for each case in a TB cluster. We evaluated LITT using a diverse dataset of 534 cases in 56 clusters (size range: 2-69 cases), which were investigated locally in three different U.S. jurisdictions. Investigators and LITT agreed on the most likely source case for 145 (80%) of 181 cases. By reviewing discrepancies, we found that many of the remaining differences resulted from errors in the dataset used for the LITT algorithm. In addition, we developed a graphical user interface, user's manual, and training resources to improve LITT accessibility for frontline staff. While LITT cannot replace thorough field investigation, the algorithm can help investigators systematically analyze and interpret complex data over the course of a TB cluster investigation. Code available at: https://github.com/CDCgov/TB_molecular_epidemiology/tree/1.0; https://zenodo.org/badge/latestdoi/166261171.

Mutation of Mycobacterium tuberculosis and Implications for Using Whole-Genome Sequencing for Investigating Recent Tuberculosis Transmission.

Tuberculosis (TB) control programs use whole-genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) for detecting and investigating TB case clusters. Existence of few genomic differences between Mtb isolates might indicate TB cases are the result of recent transmission. However, the variable and sometimes long duration of latent infection, combined with uncertainty in the Mtb mutation rate during latency, can complicate interpretation of WGS results. To estimate the association between infection duration and single nucleotide polymorphism (SNP) accumulation in the Mtb genome, we first analyzed pairwise SNP differences among TB cases from Los Angeles County, California, with strong epidemiologic links. We found that SNP distance alone was insufficient for concluding that cases are linked through recent transmission. Second, we describe a well-characterized cluster of TB cases in California to illustrate the role of genomic data in conclusions regarding recent transmission. Longer presumed latent periods were inconsistently associated with larger SNP differences. Our analyses suggest that WGS alone cannot be used to definitively determine that a case is attributable to recent transmission. Methods for integrating clinical, epidemiologic, and genomic data can guide conclusions regarding the likelihood of recent transmission, providing local public health practitioners with better tools for monitoring and investigating TB transmission.

Validation of Novel Mycobacterium tuberculosis Isoniazid Resistance Mutations Not Detectable by Common Molecular Tests.

Resistance to the first-line antituberculosis (TB) drug isoniazid (INH) is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole-genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a MIC of ≥0.25 µg/ml and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a useful tool for detecting uncommon INH resistance mutations that would otherwise be missed by current targeted molecular testing methods and suggest that its use (or use of expanded conventional or next-generation-based targeted sequencing) may provide earlier diagnosis of INH-R TB.

Statistical Method to Detect Tuberculosis Outbreaks among Endemic Clusters in a Low-Incidence Setting.

We previously reported use of genotype surveillance data to predict outbreaks among incident tuberculosis clusters. We propose a method to detect possible outbreaks among endemic tuberculosis clusters. We detected 15 possible outbreaks, of which 10 had epidemiologic data or whole-genome sequencing results. Eight outbreaks were corroborated.

Representativeness of Tuberculosis Genotyping Surveillance in the United States, 2009-2010.

Genotyping of Mycobacterium tuberculosis isolates contributes to tuberculosis (TB) control through detection of possible outbreaks. However, 20% of U.S. cases do not have an isolate for testing, and 10% of cases with isolates do not have a genotype reported. TB outbreaks in populations with incomplete genotyping data might be missed by genotyping-based outbreak detection. Therefore, we assessed the representativeness of TB genotyping data by comparing characteristics of cases reported during January 1, 2009-December 31, 2010, that had a genotype result with those cases that did not. Of 22,476 cases, 14,922 (66%) had a genotype result. Cases without genotype results were more likely to be patients <19 years of age, with unknown HIV status, of female sex, U.S.-born, and with no recent history of homelessness or substance abuse. Although cases with a genotype result are largely representative of all reported U.S. TB cases, outbreak detection methods that rely solely on genotyping data may underestimate TB transmission among certain groups.

Molecular epidemiology of Mycobacterium tuberculosis in the United States-Affiliated Pacific Islands.

The United States-Affiliated Pacific Islands (USAPI) are part of the US National Tuberculosis (TB) Surveillance System and use laboratory services contracted through a cooperative agreement with the Centers for Disease Control and Prevention (CDC). In 2004, the CDC established the National Tuberculosis Genotyping Service, a system to genotype 1 isolate from each culture-confirmed case of TB. To describe the molecular epidemiology of TB in the region, we examined all Mycobacterium tuberculosis isolates submitted for genotyping from January 1, 2004, to December 31, 2008. Over this time period, the USAPI jurisdictions reported 1339 verified TB cases to the National Tuberculosis Surveillance System. Among 419 (31%) reported culture-confirmed TB cases, 352 (84%) had complete genotype results. Routine TB genotyping allowed, for the first time, an exploration of the molecular epidemiology of TB in the USAPI.

TB-Lineage: an online tool for classification and analysis of strains of Mycobacterium tuberculosis complex.

This paper formulates a set of rules to classify genotypes of the Mycobacterium tuberculosis complex (MTBC) into major lineages using spoligotypes and MIRU-VNTR results. The rules synthesize prior literature that characterizes lineages by spacer deletions and variations in the number of repeats seen at locus MIRU24 (alias VNTR2687). A tool that efficiently and accurately implements this rule base is now freely available at http://tbinsight.cs.rpi.edu/run_tb_lineage.html. When MIRU24 data is not available, the system utilizes predictions made by a Naïve Bayes classifier based on spoligotype data. This website also provides a tool to generate spoligoforests in order to visualize the genetic diversity and relatedness of genotypes and their associated lineages. A detailed analysis of the application of these tools on a dataset collected by the CDC consisting of 3198 distinct spoligotypes and 5430 distinct MIRU-VNTR types from 37,066 clinical isolates is presented. The tools were also tested on four other independent datasets. The accuracy of automated classification using both spoligotypes and MIRU24 is >99%, and using spoligotypes alone is >95%. This online rule-based classification technique in conjunction with genotype visualization provides a practical tool that supports surveillance of TB transmission trends and molecular epidemiological studies.

Using genotyping and geospatial scanning to estimate recent mycobacterium tuberculosis transmission, United States.

To determine the proportion of reported tuberculosis (TB) cases due to recent transmission in the United States, we conducted a cross-sectional study to examine culture-positive TB cases with complete genotype results (spoligotyping and 12-locus mycobacterial interspersed repetitive unit-variable-number tandem repeat typing) reported during January 2005-December 2009. Recently transmitted cases were defined as cases with matching results reported within statistically significant geospatial zones (identified by a spatial span statistic within a sliding 3-year window). Approximately 1 in 4 TB cases reported in the United States may be attributed to recent transmission. Groups at greatest risk for recent transmission appear to be men, persons born in the United States, members of a minority race or ethnic group, persons who abuse substances, and the homeless. Understanding transmission dynamics and establishing strategies for rapidly detecting recent transmission among these populations are essential for TB elimination in the United States.

Relationship between Mycobacterium tuberculosis phylogenetic lineage and clinical site of tuberculosis.

BACKGROUND: Genotyping of Mycobacterium tuberculosis has revealed 4 major phylogenetic lineages with differential distribution worldwide. It is not clear whether different lineages are associated with different sites of infection (eg, pulmonary tuberculosis versus extrapulmonary tuberculosis). We sought to determine whether M. tuberculosis lineage is associated with the site of tuberculosis disease. METHODS: We conducted a cross-sectional analysis of all culture-confirmed cases of tuberculosis with routinely determined M. tuberculosis spoligotype-defined lineage reported to the US National Tuberculosis Surveillance System from 2004 through 2008. Odds ratios (ORs) were used to assess the relation between disease site and M. tuberculosis lineage, after adjustment for age, sex, human immunodeficiency virus infection status, region of birth, and race/ethnicity. RESULTS: Of 53972 reported culture-positive tuberculosis cases, 32000 (59.3%) were cases of M. tuberculosis that included complete spoligotype-based data on lineage. Of these, 23844 (74.5%) were exclusively pulmonary, 5085 (15.9%) were exclusively extrapulmonary, and 3071 (9.6%) were combined pulmonary and extrapulmonary. The percentages of tuberculosis cases that were exclusively extrapulmonary differed by lineage: East Asian, 13.0%; Euro-American, 13.8%; Indo-Oceanic, 22.6%; and East-African Indian, 34.3%. Compared with East Asian lineage, the odds of exclusively extrapulmonary tuberculosis relative to exclusively pulmonary tuberculosis were greater for Euro-American (adjusted OR, 1.3; 95% confidence interval [CI], 1.1-1.4), Indo-Oceanic (adjusted OR, 1.7; 95% CI, 1.5-1.9), and East-African Indian (adjusted OR, 1.6; 95% CI, 1.4-1.9) lineages. CONCLUSIONS: Phylogenetic lineage of M. tuberculosis is associated with the site of tuberculosis disease.

Tuberculosis genotyping information management system: enhancing tuberculosis surveillance in the United States.

Molecular characterization of Mycobacterium tuberculosis complex isolates (genotyping) can be used by public health programs to more readily identify tuberculosis (TB) transmission. The Centers for Disease Control and Prevention's National Tuberculosis Genotyping Service has offered M. tuberculosis genotyping for every culture-confirmed case in the United States since 2004. The TB Genotyping Information Management System (TB GIMS), launched in March 2010, is a secure online database containing genotype results linked with case characteristics from the national TB registry for state and local TB programs to access, manage and analyze these data. As of September 2011, TB GIMS contains genotype results for 89% of all culture-positive TB cases for 2010. Over 400 users can generate local and national reports and maps using TB GIMS. Automated alerts on geospatially concentrated cases with matching genotypes that may represent outbreaks are also generated by TB GIMS. TB genotyping results are available to enhance national TB surveillance and apply genotyping results to conduct TB control activities in the United States.

Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis.

The emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

Relapse associated with active disease caused by Beijing strain of Mycobacterium tuberculosis.

The role of microbial factors in outcomes of tuberculosis treatment has not been well studied. We performed a case-control study to evaluate the association between a Beijing strain and tuberculosis treatment outcomes. Isolates from patients with culture-positive treatment failure (n = 8) or relapse (n = 54) were compared with isolates from randomly selected controls (n = 296) by using spoligotyping. Patients with Beijing strains had a higher risk for relapse (odds ratio [OR] 2.0, 95% confidence interval [CI] 1.0-4.0, p = 0.04) but not for treatment failure. Adjustment for factors previously associated with relapse had little effect on the association between Beijing strains and relapse. Beijing strains were strongly associated with relapse among Asian-Pacific Islanders (OR 11, 95% CI 1.1-108, p = 0.04). Active disease caused by a Beijing strain was associated with increased risk for relapse, particularly among Asian-Pacific Islanders.

Human tuberculosis due to Mycobacterium bovis in the United States, 1995-2005.

BACKGROUND: Understanding the epidemiology of human Mycobacterium bovis tuberculosis (TB) in the United States is imperative; this disease can be foodborne or airborne, and current US control strategies are focused on TB due to Mycobacterium tuberculosis and airborne transmission. The National TB Genotyping Service's work has allowed systematic identification of M. tuberculosis-complex isolates and enabled the first US-wide study of M. bovis TB. METHODS: Results of spacer oligonucleotide and mycobacterial interspersed repetitive units typing were linked to corresponding national surveillance data for TB cases reported for the period 2004-2005 and select cases for the period 1995-2003. We also used National TB Genotyping Service data to evaluate the traditional antituberculous drug resistance-based case definition of M. bovis TB. RESULTS: Isolates from 165 (1.4%) of 11,860 linked cases were identified as M. bovis. Patients who were not born in the United States, Hispanic patients, patients <15 years of age, patients reported to be HIV infected, and patients with extrapulmonary disease each had increased adjusted odds of having M. bovis versus M. tuberculosis TB. Most US-born, Hispanic patients with TB due to M. bovis (29 [90.6%] of 32) had extrapulmonary disease, and their overall median age was 9.5 years. The National TB Genotyping Service's data indicated that the pyrazinamide-based case definition's sensitivity was 82.5% (95% confidence interval; 75.3%-87.9%) and that data identified 14 errors in pyrazinamide-susceptibility testing or reporting. CONCLUSIONS: The prevalence of extrapulmonary disease in the young, US-born Hispanic population suggests recent transmission of M. bovis, possibly related to foodborne exposure. Because of its significantly different epidemiologic profile, compared with that of M. tuberculosis TB, we recommend routine surveillance of M. bovis TB. Routine surveillance and an improved understanding of M. bovis TB transmission dynamics would help direct the development of additional control measures.

Tuberculosis genotyping in six low-incidence States, 2000-2003.

BACKGROUND: As tuberculosis incidence declines in the United States, a new tool for TB control efforts is Mycobacterium tuberculosis genotyping. Colorado, Iowa, Montana, New Hampshire, West Virginia, and Wisconsin began routine genotyping of all culture-confirmed TB cases in October 2000. METHODS: M. tuberculosis isolates from cases reported October 2000 through December 2003 were genotyped by spoligotyping, mycobacterial interspersed repetitive units, and IS6110-based restriction fragment length polymorphism methods. Genotyping results were linked to demographic variables from national surveillance records. Patients who were in genotype clusters were interviewed and their records reviewed to determine possible transmission links among clustered patients. Final analysis was completed during April 2004 through June 2005. RESULTS: Of 971 reported TB cases, 774 (80%) were culture-confirmed, of which 728 (94%) were genotyped. Most genotyped isolates (634 [87%]) were unique. Within 36 clusters linking 94 individuals, four clusters involved both U.S.- and foreign-born individuals. For eight clusters, genotyping results led to the discovery of previously unsuspected transmission. Transmission links between individuals were established in 21 (58%) of the 36 clusters. CONCLUSIONS: In these six low-incidence states, most isolates had unique genotypes, suggesting that most cases arose from activation of latent infection. Few TB clusters involved the foreign-born. For 58% of genotype clusters, epidemiologic investigation ascertained that clustering represented recent M. tuberculosis transmission.