Trends of Mycobacterium bovis Isolation and First-Line Anti-tuberculosis Drug Susceptibility Profile: A Fifteen-Year Laboratory-Based Surveillance

Background Mycobacterium tuberculosis causes the majority of tuberculosis (TB) cases in humans; however, in developing countries, human TB caused by M. bovis may be frequent but undetected. Human TB caused by M. bovis is considered a zoonosis; transmission is mainly through consumption of unpasteurized dairy products, and it is less frequently attributed to animal-to-human or human-to-human contact. We describe the trends of M. bovis isolation from human samples and first-line drug susceptibility during a 15-year period in a referral laboratory located in a tertiary care hospital in Mexico City. Methodology/Principal Findings Data on mycobacterial isolates from human clinical samples were retrieved from the laboratory’s database for the 2000–2014 period. Susceptibility to first-line drugs: rifampin, isoniazid, streptomycin (STR) and ethambutol was determined. We identified 1,165 isolates, 73.7% were M. tuberculosis and 26.2%, M. bovis. Among pulmonary samples, 16.6% were M. bovis. The proportion of M. bovis isolates significantly increased from 7.8% in 2000 to 28.4% in 2014 (X 2 trend, p<0.001). Primary STR resistance was higher among M. bovis compared with M. tuberculosis isolates (10.9% vs.3.4%, p<0.001). Secondary multidrug resistance (MDR) rates were 38.5% and 34.4% for M. bovis and M. tuberculosis, respectively (p = 0.637). A rising trend of primary STR monoresistance was observed for both species (3.4% in 2000–2004 vs. 7.6% in 2010–2014; p = 0.02). Conclusions/Significance There is a high prevalence and a rising trend of M. bovis isolates in our region. The proportion of pulmonary M. bovis isolates is higher than in previous reports. Additionally, we report high rates of primary anti-tuberculosis resistance and secondary MDR in both M. tuberculosis and M. bovis. This is one of the largest reports on drug susceptibility of M. bovis from human samples and shows a significant proportion of first-line anti-tuberculosis drug resistance.


Introduction
Tuberculosis (TB) remains an important health problem in several regions of the world, especially in countries with a high prevalence of HIV infection. Mycobacterium tuberculosis complex (MTBC) includes closely related species among which M. tuberculosis, M. bovis, and M. africanum are the most frequently associated with human disease. [1] Unlike M. tuberculosis, M. bovis can infect a broad range of mammals, including cattle and, therefore, it is considered a zoonosis. The main mechanism of contagion in humans is the consumption of unpasteurized dairy products and, less frequently, animal-to-human and human-to-human contact. [2][3][4] Historically, the burden of human TB caused by M. bovis (HTBMb) has been closely related to that of bovine TB (BTB) in the same region. [5] Unfortunately, data from most developing countries, where there is still inappropriate BTB control, is scarce. [6,7] There are several factors that explain the underreporting of HTBMb in these regions. First, control programs rely on acid-fast bacilli (AFB) smears as the primary diagnostic method in suspected TB cases; and mycobacterial culture is performed only when drug resistance is suspected, or treatment is failing. Second, most laboratories use culture medium containing glycerol, and this reduces the probability of M. bovis isolation. Lastly, only a few laboratories can identify MTBC at the species level. [8,9] In Mexico, one national reference laboratory (Instituto Nacional de Diagnóstico y Referencia Epidemiológicos) and 32 public health laboratories are capable of performing mycobacterial cultures. However, species-level identification is not routinely performed.
Consequently, data regarding first-line anti-tuberculosis drug susceptibility for MBTC are scarce. Therefore, describing the burden of HTBMb and first-line drug susceptibility pattern has important implications for treatment, referral, and public health policies in developing countries. We describe the trends of M. bovis isolation and first-line drug susceptibility for a referral laboratory in a tertiary care hospital during a 15-year period.

Methods
This study was conducted at Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, one of the National Institutes of Health in Mexico. This tertiary care referral center accepts adult patients from all over the country. The patient population involves complex medical and surgical cases including rheumatologic patients, bone marrow and solid organ transplant recipients, and HIV patients. The Laboratory of Clinical Microbiology receives local and referred clinical samples from other National Institutes of Health as well as other hospitals in Mexico City and nearby states for mycobacterial culture. Since 1992, as a regular practice, samples from patients suspected to have TB undergo mycobacterial culture, species-level identification and first-line drug susceptibility testing in all instances. We selected the 2000-2014 period because laboratory practices for mycobacterial isolation, identification, and susceptibility testing became more uniform then.

Ethics statement
Institutional approval for this study was obtained from the Comité Institucional de Investigación Biomédica en Humanos. All data analyzed were anonymized.

Data collection and definitions
A search was performed using the Laboratory of Clinical Microbiology database to investigate all MTBC isolates from January 2000 to December 2014. This database includes both local and referred samples. Only the first isolate was considered for the analysis when multiple samples within a 6-month period were available for a single patient. Whenever there were additional isolates from the same patient 6 months apart, they were considered as separate episodes and included for analysis.
Data on mycobacterial species, drug susceptibility, sample source, treatment, and referral status were obtained. HIV status was obtained for local samples only. Primary resistance (new cases) was defined as those samples from patients without previous anti-tuberculosis treatment. Secondary resistance (treated cases) was defined as those samples from patients who had previously received any anti-tuberculosis treatment. Resistance to both isoniazid (INH) and rifampin (RIF) was defined as multidrug resistance (MDR). Polydrug resistance was defined as resistance to two or more drugs but excluding those classified as MDR. Samples from sputum, bronchoalveolar lavage, endotracheal aspirate, gastric aspirate, lung and pleural biopsy, and pleural fluid were classified as pulmonary. Liver, spleen, and gastrointestinal biopsies, as well as ascites fluid, and fecal samples were classified as abdominal. Additionally, if samples from the same patient were obtained from a pulmonary and an extrapulmonary source, the case was classified as pulmonary.

Microbiology procedures
As a laboratory standard procedure, all samples obtained from bronchoalveolar lavage, cerebrospinal fluid, and biopsies from any tissue or abscess were cultured in mycobacteria-specific culture medium, regardless of TB suspicion. Sputum and urine samples were cultured for mycobacteria at the request of the treating physician.
Samples were digested and decontaminated by the NALC-NaOH method as previously described. [10,11] After digestion, samples were inoculated in both Löwenstein-Jensen medium and MGIT tubes (Becton-Dickinson, Sparks, MA, USA) according to the manufacturer's specifications. Biopsy samples were additionally inoculated in Stonebrink culture medium. Additionally, smears from all samples were prepared for Ziehl-Neelsen and Auramine-rhodamine stain. Isolates obtained from MGIT tubes were sub-cultured in Stonebrink and Löwenstein-Jensen medium. All positive cultures were further identified by DNA probe (Accuprobe, GEN-PROBE, San Diego, CA). Biochemical tests (niacin production, nitrate reduction, thiophen-2-carboxylic acid anhydride susceptibility, and pyrazinamidase deamidation) for the identification of M. bovis were performed in those positive cultures with dysgonic growth. [12] Spoligotyping was performed for local isolates as previously described, and data was entered into an international database (www.mbovis.org). [13] Susceptibility testing for anti-tuberculosis drugs was performed for INH, RIF, streptomycin (STR) and ethambutol (EMB). For this purpose, and according to the manufacturer's specifications, the radiometric BACTEC 460 TB culture system (Becton-Dickinson, Sparks, MA, USA) was used from the years 2000 to 2010 with the following drug concentrations: INH (0.1 μg/mL), RIF (2.0 μg/mL), EMB (7.5 μg/mL), and STR (6.0 μg/mL); then, from 2010 on, the BACTEC MGIT 960 culture system (Becton-Dickinson, Sparks, MA, USA) was used with the following concentrations: INH (0.1 μg/mL), RIF (1.0 μg/mL), EMB (5.0 μg/mL), STR (2.0 μg/mL). This laboratory is subjected to regular quality control evaluations by the Centers for Disease Control and Prevention, and the College of American Pathologists for identification and susceptibility testing of mycobacteria species.

Statistical analysis
Statistical analysis was performed using STATA 11.0 software (StataCorp, College Station, TX, USA). Categorical data was summarized using frequency tables, and the X 2 test was used for comparison between groups. The M. bovis case proportion by year was analyzed obtaining a X 2 for trend by the Armitage test (regression). A p-value <0.05 was determined as statistically significant for all tests.

Mycobacterial species and patient characteristics
During the study period, 81,521 samples were processed for mycobacterial culture (Fig 1).  Fig 2). Spoligotype pattern was available for 63.5% (108/170) of the local samples (S1 Table).

Susceptibility to first-line anti-tuberculosis drugs
Data on first-line anti-tuberculosis drugs susceptibility were available for 1,139 (97.7%) isolates (Table 2). When considering monoresistance among all isolates, 10.9% of M. bovis and 3.2% of M. tuberculosis were resistant to STR (p<0.001). This association remained after stratifying by new and treated cases (p<0.001 and p = 0.032, respectively). Total MDR among all cases was 11.9% for M. tuberculosis and 7.6% for M. bovis (p = 0.038). This same association was observed among new cases (6.8% vs. 3%, p = 0.026). However, among treated cases no difference was observed.

Discussion
This report demonstrates a high prevalence and a rising trend in the proportion of M. bovis isolation in our laboratory. This report is one of the largest on first-line anti-tuberculosis drug profile of M. bovis and shows a noteworthy proportion of first-line anti-tuberculosis drug resistance and secondary MDR isolates.
The proportion of M. bovis isolates in this study (26.2%) is much higher than that reported by other hospital-based studies in Latin America (0.4%) and by other hospitals in Mexico (<1%). [14,15] This may be explained by the larger study period of the present report and the high proportion of samples from immunosuppressed patients who are at a greater risk for M. bovis infection, as documented in previous studies. [16] In fact, isolates obtained from HIVinfected patients accounted for 19.2% of the local samples. [17] We also identified a rising trend in the proportion of cases caused by M. bovis across time. HMBTb is considered a reflection of the BTB burden in the region. In fact, we recently identified a high burden of bovine and human TB in a dairy production facility in rural Mexico. [2] This also correlates with previous reports of M. bovis among artisanal dairy products, which have been linked to HMBTb cases in Mexico and along the south border cities of the United States. [18,19] Mexico is considered a country of "sporadic occurrence" of BTB by the World Organization for Animal Health. However, like in many countries in the region, the test-and-slaughter strategy for bovine tuberculosis control is not universally implemented. [6] The main obstacles for BTB control in Mexico are financial and cultural. Official government data reports an overall prevalence of BTB of 2.05% in 2015, but it reaches 16.5% among dairy farms. [20] The reason for this difference may be explained by the fact that meat producing regions require to be certified as BTB free for cattle export. On the contrary, in dairy production farms, BTB only mildly affects production and pasteurization eliminates M. bovis. [21] Unfortunately, about 30% of the milk production in Mexico is sold without pasteurization, mostly to small retailers and artisan cheese producers. [22] The respiratory route of contagion is considered less efficient for M. bovis than for M. tuberculosis. However, recent data detailing outbreaks in the community and hospitals demonstrated that human-to-human contagion is not as unlikely as previously believed. [3,23,24] Interestingly, we observed an important proportion (16.6%) of M. bovis isolates recovered from pulmonary samples from a mainly urban population. Therefore, it may be hypothesized that airborne human-to-human transmission of M. bovis may occur in the community, but remains undetected in our region given that mycobacterial culture is not routinely performed. Unfortunately, as an important limitation of this study, the lack of clinical and epidemiologic data precludes us from reaching a definite conclusion.   We also report a high rate of first-line anti-tuberculosis resistance for MBTC. The proportion of INH (14%), RIF (7.6%), and STR (5.5%) primary resistance found among new cases is considerably higher for INH and RIF than in previous reports from our group in 1995 (INH 6%, RIF 2%, STR 6%). [25] These proportions are also higher than those from other reports in Mexico (1995 to 2006) among new cases (INH 9%, RIF 3%, and MDR 4.5%), and are also higher than recent data from the National Survey on TB Drug Resistance in Mexico (INH 3.5%, RIF 0.1%, STR 4% and MDR 2.3%). [26,27] This discrepancy may be explained by the different periods from which data was collected and the dissimilar patient population (ours being hospital-based and including more HIV-infected, and immunosuppressed patients).
When comparing the resistance profiles of M. bovis and M. tuberculosis, we observed a considerably higher primary MDR M. tuberculosis proportion, and a significantly higher STR monoresistance among M. bovis isolates. Data regarding drug susceptibility for M. bovis TB in It has been suggested that HTBMb cases may be at a higher risk for developing MDR strains if natural resistance to pyrazinamide is not considered and monoresistance to INH or RIF is present. [38,39] We identified a similar proportion of secondary MDR M. bovis and M. tuberculosis isolates. Unfortunately, only a few cases were analyzed and no data on previous treatments or outcomes were available, therefore we are unable to conclude if this assumption is true. Nevertheless, it should be recognized that TB cases caused by MDR M. bovis may result in disease that is harder to treat on a second-line drug regimen. This highlights the need for performing species-level identification and drug susceptibility testing whenever M. bovis is suspected.
In conclusion, we believe that data contained in this study is relevant in terms of public health and highlights the need for more stringent control of BTB in our country. It also underscores the importance of proper identification of M. bovis given that the considerable rate of primary resistance to INH and RIF along with the natural pyrazinamide resistance may result in treatment failures and select for MDR strains.
Supporting Information S1