Epidemiology of Human Mycobacterium bovis Disease, California, USA, 2003–2011

Disease was associated with the Hispanic binational population and immunosuppressive conditions, including diabetes.

M ycobacterium bovis, part of the Mycobacterium tuberculosis complex, is a zoonotic pathogen that can cause tuberculosis (TB) disease in a broad range of mammalian hosts (1). TB disease caused by M. bovis is clinically, radiographically, and pathologically indistinguishable from TB caused by M. tuberculosis (2). M. bovis transmission to humans most frequently occurs through consumption of unpasteurized, contaminated dairy products, but person-to-person transmission has been reported (3,4). The consumption of contaminated unpasteurized dairy products has been suggested as a major contributor to human M. bovis disease for several reasons: 1) the near absence of M. bovis disease among infants <12 months of age; 2) a high percentage of extrapulmonary disease, particularly abdominal disease, among patients with M. bovis disease; and 3) an association between positive interferon-γ release assay results and consumption of unpasteurized dairy products (5)(6)(7)(8)(9)(10)(11).
In the United States, 1%-2% of all human TB cases are attributable to M. bovis infection (7), but in certain geographic regions and communities, human M. bovis infection accounts for a much higher percentage of the cases. During 2001-2005, M. bovis accounted for nearly 10% of culture-positive TB isolates in San Diego, California, USA, including 54% of those from children (<15 years of age) and 8% of those from adults (>15 years of age) (8). Nearly all (97%) case-patients with M. bovis disease were among the Hispanic population, and 60% of those case-patients were born in Mexico (8). During 2001During -2004, an investigation in New York, New York, USA, showed a high prevalence of TB caused by M. bovis among the Hispanic community. New York investigators reported that 57% of M. bovis case-patients were born in Mexico, and 83% of the interviewed case-patients consumed unpasteurized cheeses produced in Mexico while living in the United States (12).
The internationally recognized genotypic method for identifying M. bovis is spacer oligonucleotide typing (13). In 2004, the CDC (Centers for Disease Control and Prevention) Tuberculosis Genotyping Program (now called the National Tuberculosis Genotyping Service, http:// www.cdc.gov/tb/publications/factsheets/statistics/genotyping.htm) began spoligotyping M. tuberculosis complex isolates from US patients with culture-positive TB (14). In California, the percentage of culture-positive isolates spoligotyped each year has gradually increased from 35% in 2004 to 92% in 2011. The incompleteness and variability (by geographic location) of spoligotype testing over this period exclude trend analysis and populationbased M. bovis studies using this genotypic method. However, pyrazinamide monoresistance can serve as a proxy measure for M. bovis because M. bovis is intrinsically resistant to pyrazinamide but pyrazinamide monoresistance is rare among M. tuberculosis isolates (7,15,16). A recent national study on pyrazinamide resistance showed that 0.7% (196/27,428) M. tuberculosis isolates were pyrazinamide monoresistant (15). Since 2003, ≈97% of all culture-positive TB isolates in California have had initial (i.e., pretreatment) drug susceptibility testing for pyrazinamide, isoniazid, and rifampin.
M. bovis disease is of particular concern because of the high percentage of cases among children and because of its association with zoonotic and foodborne transmission, HIV co-infection, and poor treatment outcomes compared with M. tuberculosis disease (6,(17)(18)(19). Further investigation into M. bovis disease is needed to understand the epidemiology of cases among children and adults, its association with immunosuppressive conditions, and the association of those conditions with treatment outcomes. We conducted a retrospective review of California TB surveillance data to evaluate trends for TB cases attributable to M. bovis, evaluate epidemiologic differences between M. bovis TB cases in adults and children, and identify risk factors associated with M. bovis disease compared with M. tuberculosis disease. We also conducted an evaluation of the accuracy of pyrazinamide monoresistance as a proxy measure for M. bovis disease by using surveillance and genotyping data.

Methods
The study population included all patients with cultureconfirmed TB reported to the California TB registry during 2003-2011 and who had initial drug susceptibility testing results for isoniazid, rifampin, and pyrazinamide. Patients were classified as having M. bovis disease if the initial drug susceptibility results indicated resistance to pyrazinamide and susceptibility to isoniazid and rifampin. Sociodemographic, clinical, and treatment outcome information for all case-patients was abstracted from the TB registry. TB case registry data were matched to the California HIV/ AIDS registry to identify HIV co-infection status. This analysis was conducted as part of the California Department of Public Health's mandate to routinely collect and analyze surveillance data for public health purposes. The CDC (Atlanta, GA, USA) determined that the project was not human subject research and did not require approval by an institutional review board.
To compare the differences between child and adult populations, we stratified M. bovis case-patients by their age at the time TB was reported. The child population consisted of patients <15 years of age, and the adult population consisted of patients >15 years of age. To evaluate characteristics associated with TB disease caused by M. bovis compared with TB disease caused by M. tuberculosis, we conducted bivariate analysis with variables that were added to the national TB surveillance system in 2010. These variables include birth country of parents/ guardians (for child case-patients), primary reason evaluated for TB disease, diabetes mellitus, and other immunosuppressive conditions. Immunosuppressive conditions, excluding HIV co-infection and diabetes mellitus, comprised end-stage renal disease, anti-tumor necrosis factor-α therapy-associated immunosuppression, solid organ transplant-associated immunosuppression, or other immunosuppressive condition as indicated by medical records or a health care provider. In addition, patient variables previously shown to be associated with TB disease caused by M. bovis (i.e., country of birth, race/ethnicity, age, HIV co-infection, site of disease, and death before treatment completion) were included in the bivariate analysis (7,19). Sociodemographic and clinical variables shown to be significantly associated with M. bovis disease at the bivariate level were put into a logistic regression model. The final logistic regression model was constructed by using the backward stepwise elimination procedure, removing predictors with p>0.05.
For TB cases during 2004-2011, the sensitivity and positive predictive value of the pyrazinamide monoresistance case definition were calculated by using spoligotyping data as the reference standard. The signature spoligotypes differentiating M. bovis and M. tuberculosis have been described previously in detail (20). Spoligotyping was conducted at the California Microbial Diseases Laboratory (Richmond, CA, USA), and the resulting data were entered into the TB Genotyping Information Management System (http://www.cdc.gov/tb/programs/genotyping/tbgims/default.htm). The TB registry and TB Genotyping Information Management System databases were merged by patients' unique case numbers after spoligotyping data were deduplicated. Spoligotyping results indicating a genotype other than M. bovis or M. tuberculosis (including M. bovis bacillus Calmette-Guérin (BCG) strains) and isolates missing spoligotyping data or initial drug susceptibility testing results for pyrazinamide, isoniazid, or rifampin were excluded from analysis.
Analyses were conducted by using SAS version 9.3 (SAS Institute, Cary, NC, USA). Separate Poisson regression models were used to identify temporal trends in the incidence of TB caused by M. tuberculosis and M. bovis.
Year of TB case report was used as the explanatory variable, TB case number as the dependent variable, and state population size as the offset variable. Population denominators were obtained from the US Census Bureau's current population survey (21). Trends of the annual percentage of TB cases attributable to M. bovis were examined by using the Cochran-Armitage trend test. Epidemiologic differences between M. bovis and M. tuberculosis were compared by using the χ 2 test or Fisher exact test. Differences in median time to completion of therapy from start of therapy were analyzed by using the Wilcoxon rank-sum test. Trends and differences in disease characteristics were considered statistically significant if p<0.05 (2-sided).

Burden and Trends
During 2003-2011, a total of 24,424 verified TB cases were reported in California. Of these, 5,061 (21.0%) culture-negative cases were excluded ( Figure 1). Approximately 3.0% (611/19,363) of culture-positive cases were excluded because results of initial drug susceptibility testing for isoniazid, rifampin, or pyrazinamide were absent. M. bovis was identified by pyrazinamide monoresistance  Figure 3). The annual percentage of TB cases attributable to M. bovis among the child population did not change significantly (p = 0.15), but the percentage among adults increased from 3.0% to 5.5% (p<0.001).

Epidemiology of M. bovis Disease in Children versus Adults
Several differences were observed between child and adult case-patients with M. bovis disease (Table 1). Child casepatients were more likely to be Hispanic and US-born and to have extrapulmonary disease. All M. bovis case-patients with concurrent HIV co-infection were adults. During 2003-2011, a total of 11.4% (80/699) of all case-patients with M. bovis disease died before treatment was completed. Among children, 1.2% (1/82) died before treatment was completed, compared with 12.8% (79/617) of adults (p = 0.002). Among case-patients who completed treatment, the median time to completion did not differ significantly between children and adults.

M. bovis Epidemiology versus M. tuberculosis Epidemiology
Bivariate analysis showed that the following patient characteristics were associated with TB caused by M. bovis but not by TB caused by M. tuberculosis: Hispanic ethnicity, birth in Mexico, incidental laboratory result as primary reason for TB evaluation, HIV co-infection, presence of extrapulmonary TB disease, and diabetes and other immunosuppressive conditions. Results were similar when we repeated the bivariate analysis using the spoligotyping method to identify M. bovis disease. A higher percentage of M. bovisthan M. tuberculosis-infected patients had diabetes; however, this association was no longer significant (p = 0.08) because the study population was smaller. The association between M. bovis disease and other immunosuppressive conditions remained significant (p = 0.002).
During 2010-2011, case-patients with M. bovis disease were more likely than those with M. tuberculosis disease to die before treatment completion (15.8% vs. 8.6%, p = 0.006) ( Table 2). Among case-patients who died before treatment completion, those with M. bovis disease were more likely than those with M. tuberculosis disease to have had >1 concurrent immunosuppressive condition (73% vs. 53%, p = 0.05). Of the 26 M. bovis case-patients who died before treatment completion, 19 (73%) had >1 concurrent immunosuppressive condition: diabetes and end-stage renal disease (n = 6), diabetes (n = 5), HIV co-infection (n = 1), end-stage renal disease (n = 1), post-organ transplantation-associated immunosuppression (n = 1), end-stage renal disease and post-organ transplantation-associated immunosuppression (n = 1), or any other immunosuppressive condition (n = 4).   conditions were independently associated with M. bovis disease compared with M. tuberculosis disease (  in the United States often results from consumption of unpasteurized dairy products originating from foreign countries, including Mexico (6,12,22,23). National pasteurization requirements, strict regulations on the importation of dairy cattle from Mexico into the United States, and an effective US bovine TB eradication program have substantially reduced M. bovis infection in US-born cattle (22,24). In the past century, the prevalence of M. bovis disease in US dairy herds has decreased from ≈5.0% to <0.001% (25 (1). §Of case-patients with any pulmonary involvement. ¶Includes end-stage renal disease, immunosuppression associated with receiving anti-tumor necrosis factor-α therapy, immunosuppression associated with receiving solid-organ transplant, or other immunosuppressive conditions, but excludes diabetes and HIV co-infection.
Diego have been found to be genetically related to M. bovis strains from cattle in Mexico (22). Expansion of M. bovis disease surveillance and genotyping to include whole-genome sequencing may add discriminatory power beyond traditional genotyping and help to identify the source and route of transmission of M. bovis (26). Sharing of wholegenome sequencing data between countries and different health agencies may enhance future national and international prevention interventions. Despite the relatively high percentage of extrapulmonary disease, 57% of M. bovis case-patients in our study had pulmonary involvement; this percentage is consistent with findings in other studies in the United States (7,8). Person-to-person transmission of M. bovis is considered infrequent, but the magnitude of such transmission has not been precisely quantified (27). Our findings are consistent with those from past research, which has shown that case-patients with M. bovis and M. tuberculosis pulmonary disease do not differ significantly in 2 of the key indicators of infectivity: presence of lung cavitation on chest radiographs and presence of acid-fast bacilli in sputum smears (6,7,28). In addition, previous research from pulmonary TB contact investigations showed that TB infection conversion rates among contacts did not differ significantly by mycobacterial species of the source case, suggesting that M. bovis is equally as transmissible as M. tuberculosis (28). However, current TB contact investigation guidelines do not include risk factors for M. bovis transmission. Although it is recommended to prioritize immunocompromised contacts of M. tuberculosis patients for evaluation, our data suggest that it may be even more important to prioritize immunocompromised contacts of M. bovis patients during contact investigations.
In our evaluation of the definition of pyrazinamide monoresistance, we found that isolates genotyped as M. tuberculosis may have been misclassified as M. bovis because of the pyrazinamide monoresistance definition. Misclassifications occurred most notably among the non-Hispanic Asian patients and might be explained by host, environmental (e.g., regional programmatic differences in TB treatment), and bacterial characteristics. From a recent national multivariate analysis of pyrazinamide resistance, Kurbatova et al. (15) suggested that bacterial lineage, not host characteristics, was the primary association between pyrazinamide monoresistance and M. tuberculosis disease.
Despite the possible definition-associated overestimation of M. bovis disease, the results from our study may still underestimate the true burden of M. bovis disease in California. Cases in children may be underestimated in the study population because sputum or gastric aspirate specimens are not consistently obtained from young children (29). In addition, M. bovis disease may be present in casepatients in the culture-negative subpopulation. We found case-patients with culture-negative TB to be similar to case-patients with culture-confirmed M. bovis disease with respect to age, extrapulmonary disease, and Hispanic ethnicity (data not shown).
Given the limitations of traditional genotyping and surveillance, we could not assess whether the increase in the number and percentage of adult M. bovis case-patients was to the result of recently acquired TB or reactivation of a previous infection. In addition, analysis of the new variables that were added to the national TB surveillance system in 2010 was hindered by the small number of child case-patients with M. bovis disease during 2010-2011. Also, because we did not conduct a medical chart review, we could not determine the cause of death among TB case-patients who died before the completion of TB treatment. Because of limitations in the national TB surveillance report form, we were unable to assess several possible relevant risk factors, including consumption of unpasteurized dairy products and the potential protective effect of the BCG vaccine against M. bovis disease. Although there is little information in the literature on the efficacy of the BCG vaccine in protecting against human M. bovis disease, the vaccine is notably protective against extrapulmonary disease and childhood TB disease, both of which are characteristic of M. bovis disease (30)(31)(32)(33).
In summary, human M. bovis disease incidence has not declined in California, and the percentage of TB cases attributable to M. bovis has increased, exceeding the overall average for the United States. In California, there are ongoing interventions designed to limit the demand for and distribution of unpasteurized and contaminated dairy F rom the Latin bos ("ox" or "cow") Mycobacterium bovis is a virulent bacterial species originally isolated from tubercules in cattle. Robert Koch, who discovered the tubercle bacillus in 1882, believed that M. bovis was not a danger to humans. Theobald Smith and others established beyond doubt that, contrary to Koch's belief, M. bovis could infect humans but was not the usual source of human infection. In 1908, French scientists Albert Calmette and Camille Guérin chose an M. bovis strain for their work on a tuberculosis vaccine. They repeatedly subcultured the isolate on a mixture of glycerol, potato, and bile for 13 years until it was sufficiently attenuated to be used as a vaccine. The bacillus Calmette-Guérin (BCG) vaccine was adopted by the League of Nations as the standard tuberculosis vaccine in 1928 and continues to be used in most developing countries.